De novo design of protein switches

Abstract

To achieve a de novo design of a protein system which switches a conformational state in the presence of an external input.SOLUTION: Disclosed are: protein switches that can sequester bioactive peptides and / or binding domains, holding them in an inactive ("off") state, until combined with a second designed polypeptide called the key to induce a conformational change that activates ("on") the bioactive peptide or binding domain; components of such protein switches; and use thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] Cross-reference to previously filed applications This application claims priority to U.S. Provisional Patent Application No. 62 / 700,681, filed July 19, 2018, No. 62 / 785,537, filed December 27, 2018, and No. 62 / 788,398, filed January 4, 2019, each of which is incorporated by reference in its entirety.

[0002] Reference to sequence listings submitted electronically via EFS-Web The contents of the Sequence Listing submitted electronically in an ASCII text file submitted with this application (Name: 18-1054-PCT_Sequence-Listing_ST25.txt, Size: 32,278 kb, and Creation Date: July 19, 2019) are hereby incorporated by reference in their entirety. [Background technology]

[0003] Considerable progress has been made in the de novo design of stable protein structures based on the principle that proteins fold into the lowest free energy state. These efforts have focused on maximizing the free energy gap between the desired folded structure and all other structures. Designing proteins that can switch shape is more challenging, because the states to exist must have sufficiently low free energy compared to the unfolded state, and the free energy difference between the states must be small enough that the occupancy of the states can be switched by an external input. The de novo design of protein systems that switch shape states in the presence of an external input has not been achieved. Summary of the Invention

[0004] In a first aspect, the disclosed non-naturally occurring polypeptide comprises: (a) A helical bundle containing 2–7 alpha-helices; (b) an amino acid linker connecting each alpha helix.

[0005] In one embodiment, each helix is ​​independently 18-60, 18-55, 18-50, 18-45, 22-60, 22-55, 22-50, 22-45, 25-60, 25-55, 25-50, 25-45, 28-60, 28-55, 28-50, 28-45, 32-60, 32-55, 32-50, 32-45, 35-60, 35-55, 35-50, 35-45, 38-60, 38-55, 38-50, 38-45, 40-60, 40-58, 40-55, 40-50, or 40-45 amino acids in length. In another embodiment, each amino acid linker is independently between 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 2-9, 3-9, 4-9, 5-9, 6-9, 7-9, 8-9, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 2-7, 3-7, 4-7, 5-7, 6-7, 2-6, 3-6, 4-6, 5-6, 2-5, 3-5, 4-5, 2-4, 3-4, 2-3, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length without any additional functional sequences that may be fused to the linker. In further embodiments, the polypeptide comprises one or more bioactive peptides in at least one of the alpha helices, wherein the one or more bioactive peptides are capable of selectively binding to a defined target, and the one or more bioactive peptides may comprise one or more bioactive peptides selected from the non-limiting group consisting of SEQ ID NOs: 60, 62-64, 66, 27052-27093, and 27118-27119.

[0006] In another aspect, the disclosure provides a caged polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27,278-27,321, as well as non-naturally occurring polypeptides comprising a polypeptide having at least 40% sequence identity along its length to the amino acid sequence of a caged polypeptide listed in Table 2, Table 3, and / or Table 4, wherein the N-terminal and / or C-terminal 60 amino acids of the polypeptide are optional, and the sequence identity requirement does not include the optional amino acid residues. In one aspect, the disclosure provides a non-naturally occurring polypeptide comprising a polypeptide having at least 40% sequence identity along its length to the amino acid sequence of a caged polypeptide listed in Table 2, Table 3, and / or Table 4, without including the optional amino acid residues. In one embodiment of each of these aspects, the polypeptide comprises an amino acid sequence having an amino acid sequence that has at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along its length, without including any amino acid residue, to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-91, SEQ ID NOs:1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27,278-27,321, and the caged polypeptides listed in Table 2, Table 3, and / or Table 4.

[0007] In one embodiment of any of the aspects of the present disclosure, the non-naturally occurring polypeptide further comprises one or more bioactive peptides within or replacing the latch region of the polypeptide, and the one or more bioactive peptides may comprise one or more bioactive peptides selected from the non-limiting group consisting of SEQ ID NOs: 60, 62-64, 66, 27052-27093, and 27118-27119.

[0008] In another aspect, non-naturally occurring polypeptides are provided that include a key polypeptide disclosed herein or a key polypeptide selected from the group consisting of SEQ ID NOs: 14318-26601, 26602-27015, and 27016-27051, as well as polypeptides having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along their length to the amino acid sequence of a key polypeptide listed in the table, not including any optional amino acid residues. In one embodiment, the polypeptide comprises an amino acid sequence having an amino acid sequence that has at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along its length, not including optional residues, to the amino acid sequence of a key protein selected from the group consisting of the key polypeptides listed in Table 2, Table 3, and / or Table 4.

[0009] In another embodiment, the present disclosure provides a fusion protein comprising a cage polypeptide of any embodiment or combination of embodiments of the present disclosure fused to a key polypeptide of any embodiment or combination of embodiments of the present disclosure.

[0010] In other aspects, the disclosure provides a cage polypeptide of the fusion protein of any embodiment or combination of embodiments of the disclosure, a nucleic acid encoding a key polypeptide, an expression vector comprising the nucleic acid operably linked to a promoter, and / or a host cell comprising the nucleic acid and / or the expression vector. In one embodiment, the nucleic acid or expression vector is integrated into a host cell chromosome. In another embodiment, the nucleic acid or expression vector is episomal. In another embodiment, the host cell is (a) a first nucleic acid encoding a cage polypeptide of any embodiment or combination of embodiments of the present disclosure operably linked to a first promoter; (b) a second nucleic acid encoding a key polypeptide of any embodiment or embodiment of the present disclosure operably linked to a second promoter, wherein the second nucleic acid encodes a key polypeptide capable of binding to a structural region of a cage polypeptide encoded by the first nucleic acid, and wherein binding of the key polypeptide to the structural region of the cage polypeptide induces a conformational change in the cage polypeptide.

[0011] In a further embodiment of the host cell of the present disclosure, the first nucleic acid comprises a plurality of first nucleic acids encoding a plurality of different cage polypeptides. The second nucleic acid comprises a plurality of second nucleic acids encoding a plurality of different key polypeptides, the plurality of different key polypeptides comprising one or more key polypeptides capable of binding to only a subset of the plurality of different cage polypeptides and inducing a conformational change in the subset. In another embodiment, the second nucleic acid encodes a single key polypeptide capable of binding to each different cage polypeptide and inducing a conformational change in each different cage polypeptide.

[0012] In another embodiment, the host cell comprises (a) a first nucleic acid encoding a fusion protein according to any embodiment or combination of embodiments of the present disclosure operably linked to a first promoter; (b) a second nucleic acid encoding a fusion protein according to any embodiment or combination of embodiments of the present disclosure operably linked to a second promoter; (i) a cage polypeptide encoded by a first nucleic acid is activated by a key polypeptide encoded by a second nucleic acid; (ii) the cage polypeptide encoded by the first nucleic acid is not activated by the key polypeptide encoded by the first nucleic acid; (iii) the cage polypeptide encoded by the second nucleic acid is activated by the key polypeptide encoded by the first nucleic acid; (iv) the cage polypeptide encoded by the second nucleic acid is not activated by the key polypeptide encoded by the second nucleic acid.

[0013] In another aspect, the present disclosure provides a kit, (a) one or more cage polypeptides according to any embodiment or combination of embodiments of the present disclosure; (b) one or more key polypeptides according to any embodiment or combination of embodiments of the present disclosure; (c) optionally, one or more fusion proteins according to any embodiment or combination of embodiments of the present disclosure.

[0014] In one aspect, the present disclosure provides a kit, (a) a first nucleic acid encoding one or more cage polypeptides according to any embodiment or combination of embodiments of the present disclosure; (b) a second nucleic acid encoding one or more key polypeptides according to any embodiment or combination of embodiments of the present disclosure; and (c) optionally, a third nucleic acid encoding one or more fusion proteins according to any embodiment or combination of embodiments of the present disclosure.

[0015] In various embodiments, the first nucleic acid, the second nucleic acid, and / or the third nucleic acid comprise an expression vector.

[0016] In another aspect, the present disclosure provides a LOCKR switch, (a) a cage polypeptide comprising a structural domain and a latch domain further comprising one or more biologically active peptides, wherein the structural domain interacts with the latch domain to prevent activity of the one or more biologically active peptides; (b) an optional key polypeptide that binds to the cage structure region, thereby displacing the latch region and activating one or more bioactive peptides; (c) optionally, one or more effector polypeptide(s) that bind to the one or more bioactive peptides when the one or more bioactive peptides are activated.

[0017] In one embodiment, an effector polypeptide is present, comprising an effector polypeptide that selectively binds to a biologically active peptide, including, but not limited to, Bcl2, GFP1-10, and a protease.

[0018] In various embodiments, a host cell, kit, or LOCKR according to any embodiment or combination of embodiments of the present disclosure comprises one or more cage polypeptides and one or more key polypeptides having amino acids along their length that have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, not including optional residues, to a cage polypeptide and key polypeptide, respectively, in the same row of Table 1, Table 2, Table 3, and / or Table 4, or a cage polypeptide and key polypeptide, respectively, in the same row of Table 2, Table 3, and / or Table 4. In further embodiments, the one or more biologically active peptides may include one or more biologically active peptides selected from the non-limiting group consisting of SEQ ID NOs: 60, 62-64, 66, 27052-27093, and 27118-27119.

[0019] In other embodiments, the host cell, kit, or LOCKR according to any embodiment or combination of embodiments of the present disclosure comprises: (a) one or more cage polypeptides, including a cage polypeptide selected from the group consisting of SEQ ID NOs: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27,278-27,321, and one or more cage polypeptides having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along its length to an amino acid sequence of a cage polypeptide listed in Table 2, Table 3, and / or Table 4, without any optional amino acid residues, wherein the N-terminal and / or C-terminal 60 amino acids of the polypeptide are optional; (b) one or more key peptides comprising one or more polypeptides selected from the group consisting of key polypeptides selected from the group consisting of SEQ ID NOs: 14318-26601, 26602-27015, 27016-27051, and polypeptides having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along their length to an amino acid sequence of a key polypeptide listed in Table 2, Table 3, and / or Table 4, without including any amino acid residue.

[0020] In another aspect, the disclosure provides for the use of the polypeptides, fusion proteins, nucleic acids, expression vectors, host cells, kits, and / or LOCKR switches disclosed herein to sequester bioactive polypeptides in a cage polypeptide, holding them in an inactive ("off") state until combined with a key polypeptide to induce a conformational change that activates ("on") said bioactive peptide. [Brief description of the drawings]

[0021] [Figure 1]Design of the LOCKR switch system. Figure 1A shows a thermodynamic model illustrating the design goals. The structural and latch regions in the cage form a switch with some equilibrium in the open and closed states. The key can bind to the cage to promote the open state, allowing the target to bind to the latch. Figure 1B shows plots for two values ​​of KLT from the model in (a) to show how the percentage of target bound is affected by the addition of the key (KCK = 1 nM), with different colored curves showing the effect of logarithmic reduction in Kopen = [open] / [closed]. Figure 1C shows that loops are added to the homotrimer 5L6HC3_15 to form a monomeric 5- and 6-helix framework, with the double mutant V217S / I232S weakening the latch and allowing it to be replaced by the key, resulting in a LOCKR system that can bind to exogenous keys. Figure 1D shows chemical denaturation with guanidine chloride (Gdm) monitoring the mean residue ellipticity (MRE) at 222 nm. Figure 1E shows small angle X-ray scattering (SAXS) spectra of the monomeric frameworks that closely match each other and the native homotrimer. Figure 1F shows pull-down assays showing that KEY binds to the truncated 5-helical framework and LOCKR(V217S / I232S) but not to the 6-helical monomer. Free GFP-KEY was added to the monomeric framework immobilized on a plate via the hexahistidine tag, and after a series of washing steps, binding was measured by GFP fluorescence (n=2, error bars indicate standard deviation). [Diagram 2]BimLOCKR design and activity. Figure 2A. The free energy of the latch-cage contact was tuned through suboptimal Bim-cage interactions (left, shown as altered hydrophobic packing and buried hydrogen bonds) and by exposing hydrophobic residues at the edge of the contact (right) as toeholds. B) Introduction of a toehold allows activation of 250 nM BimLOCKR by addition of 5 µM key ("on" bar) via Biolayer Interferometry. C) Biolayer Interferometry shows key-dependent binding to Bcl2 by 250 nM BimLOCKR; association from 0 to 500 s, and dissociation from 500 to 1700 s. D) Each point is the result of fitting the data with I and extracting the response at equilibrium. The curves show similar data with shorter keys, demonstrating the ability of LOCKR to modulate KCK and affect its range of activation. [Diagram 3] Design and validation of orthogonal BimLOCKR. A) Left: LOCKR in a sketchy representation. Cage with three different latches superimposed and hydrogen bond network marked by markers. Right: Hydrogen bond network across orthogonal LOCKR contacts. b) BimLOCKR binding to Bcl2 in response to cognate key in Octet. One replicate. c) Response in Octet after 500 seconds for each switch (250 nM) and key (5 μM) pair. Average of two replicates. [Figure 4] Experimentally determined X-ray crystal structures of the desymmetrized LOCKR switch designs. (A) Crystal structure of design 1-fix-short-BIM-t0 containing the encoded BIM peptide. (B) The crystal structure of design 1fix-short-noBim(AYYA)-t0 is in excellent agreement with the design model in terms of (left) backbone, (center) hydrogen bond network, and (right) hydrophobic packing; the regions of the latch where Bim and Gfp11 are encoded are indicated, and electron density maps are displayed for the network and hydrophobic cross sections (center and right). [Diagram 5]A. A LOCKR switch that can prevent split GFP11 from complementing GFP1-10 in the absence of the key. A. The crystal structure of GFP with strand 11 (pdb 2y0g) is shown. B. Crystal structure of a prototype switch with GFP11 stabilized as a helix (mesh is electron density). C. The computational design model matches the crystal structure with a root mean square deviation of 0.87 Å. D. The experimentally determined X-ray crystal structure of the designed LOCKR switch 1fix-short-GFP-t0 shows that the 11th strand of the encoded GFP (GFP11) is an alpha helix, in excellent agreement with the design model. E. GFP fluorescence is observed only in the presence of the key peptide, indicating that the switch is functional (off when the key is absent, on when the key is present). [Figure 6] GFP11-LOCKR switch designed from Figure 4, tuned to be colocalization dependent. (A) Schematic of a test system in which colocalization dependence is controlled by coupled Spycatcher™ / Spytag™ fusions. In this model, the key activates the LOCKR switch (produces fluorescence) only when fused to Spytag, and the key is colocalized to the cage (right). Adding the key alone fails to activate the LOCKR switch (middle). (B) Fluorescence data showing the colocalization dependence of the designed LOCKR switch according to the schematic in (A). Designs 1fix-latch and 1fix-short fused to Spycatcher™ show stronger activation when mixed with their cognate keys fused to Spytag™. Keys lacking Spytag™ show significantly lower activation. [Figure 7] Caged intein LOCKR switch. A LOCKR switch designed with a caged moiety encoding a VMAc intein shows good activation when mixed with sfGFP and a key designed to be fused to a VMAn intein. SDS-PAGE shows the success of the VMAc-VMAn reaction, with a band corresponding to the correct molecular weight of the expected spliced ​​protein product. [Figure 8] Multiple sequence alignment (MSA) comparing the original LOCKR_a cage scaffold design with its asymmetric (1fix-short-noBim(AYYA)-t0) and orthogonal (LOCKRb-f) designed counterparts. Only 150 of the sites (40.8%) are identical across the MSA, with a pairwise % identity of 69.4%. The latch region (C-terminal region starts at position 311 in this MSA) has very little sequence identity / similarity. (From top to bottom, SEQ ID NOs: 17, 39, 7, 8, 9, 10, 11) [Figure 9] Superposition of the crystal structure of 1fix-short-noBim(AYYA)-t0 (Figure 4B) (white) with the X-ray crystal structure of the base scaffold 5L6HC3_15 (dark) used to create LOCKRa (Figure 1) shows that the desymmetrizing mutations (variable positions shown in Figure 8 MSA) do not affect the 3D structure of the protein. The backbone RMSD between the two proteins is 0.85 Å (from a superposition of all backbone atoms between chains A). [Figure 10] GFP plate assay to find mutations in LOCKR. Different putative LOCKR constructs were attached to Ni-coated 96-well plates via 6x-His tags, KEY-GFP was added and washed extensively. The resulting fluorescence represents KEY-GFP bound to the LOCKR construct. Truncation was used as a positive control since KEY binds to the open contact. Monomer is a negative control since it does not bind to KEY. Error bars represent standard deviation of three replicates. [Figure 11]Orthogonal LOCKR GFP assays. A) In five redesigned LOCKR constructs (b to f), the latch was truncated from the 6×-His tagged cage. The corresponding key was GFP tagged. Key-cage binding was measured by Ni pulldown of the cage and measuring the resulting GFP fluorescence. Error bars are standard deviations of three replicates. B) Each full LOCKR construct that binds to the key in (a) was supplemented with a 9 residue toehold and tested for binding to all four functional keys (a to d) in a GFP pulldown assay. Error bars are standard deviations of five replicates. Key a is likely to bind promiscuously but not activate due to the pseudosymmetric generation of LOCKR from homotrimers. LOCKRb shows no binding to its own key and is attributable to the latch strength obtained from the results in (a) and Figure 3b. [Figure 12]Mad1-SIDLOCKR switch designed for KEY-dependent transcriptional repression. (a) Crystal structure of the interaction between the Mad1-SID domain (white) and the PAH2 domain (black) of the mSin3A transcriptional repressor (PDB ID: 1E91). Caging of the Mad1-SID domain should enable KEY-dependent recruitment of the transcriptional repressor mSin3A, enabling precise epigenetic regulation. (b) Designed Mad1-LOCKR switch, with cage components encoding the Mad1-SID sequence at different positions (dark grey). (c) SDS-PAGE gel showing well-purified 1fix_302_Mad1 (1), 1fix_309_Mad1 (2), and MBP_Mad1 (3). (d) Biolayer interferometry analysis of KEY-activating binding of the Mad1-LOCKR switch with purified mSin3A-PAH2 domain. MBP-Mad1 is a positive control for mSin3a-PAH2 binding. 1fix_309_Mad1(309) shows good activation when mixed with the designed Key a. 1fix_302_Mad1(302) shows very enhanced caging of the Mad1-SID domain but no activation in the presence of Key a. Kinetic assays were performed by immobilizing 0.1 μg of biotin-mSin3A-PAH2 protein onto a streptavidin biosensor chip (ForteBio). Protein cages were tested at 50 nM in the presence or absence of 500 nM Key a. [Figure 13]Demonstration of caged STREPII-tag LOCKR switches, novel 2 plus 1 and 3 plus 1 LOCKR switches. (A) Designed 2+1 (left) and 3+1 (center) LOCKR switches were designed to encode the STREPII sequence WSHPQFEK (sequence number 63). (B-D) Biolayer interferometry (Octet) data demonstrating functionality of the STREPII-LOCKR design: Anti-strep antibodies immobilized on an anti-mouse FC chip to assess binding of the STREPII tag: (B) The designed protein shows lower binding than the positive control, suggesting that STREPII is at least partially sequestered as intended. (C) Activation of designed STREPII-3plus1_Lock_3 by 3plus1_key_3: Curves are 250 nM of cage without key compared to 250 nM cage in the presence of increasing concentrations of key ranging from 121 nM to 6000 nM. (D) 250 nM STREPII-3plus1_Lock_3 in the presence of key at 370 nM, 1111 nM, and 3333 nM: 250 nM cage without key, other plots are key at the same concentrations (370 nM, 1111 nM, and 3333 nM) but without cage. In all Octet plots, the left half is the association (binding) phase and the right half is the dissociation phase. [Figure 14]The 3 plus 1 LOCKR switch activates GFP fluorescence upon expression of Key. The LOCKR switch was designed to sequester strand 11 of GFP (GFP11) in an inactive conformation using the 3 plus 1 Cage, thereby preventing reconstitution of split GFP (composed of GFP1-10 and GFP11) and resulting in fluorescence. Expression plasmids were prepared to inducibly express Cage (p15a origin of replication, spectinomycin resistance, arabinose-inducible promoter controlling expression of GFP1-10 and LOCKR-caged GFP11) and Key (colE1 origin of replication, kanamycin resistance, and IPTG-inducible promoter). Chemically competent E. coli Stellar cells (Takarabio) were transformed according to the manufacturer's protocol with either the Cage plasmid alone or both the Cage and Key plasmids. These transformants were grown overnight at 37 °C in liquid LB medium supplemented with spectinomycin (Cage only) or spectinomycin + kanamycin (Cage and Key). The resulting cultures were diluted 1 / 100 in fresh LB medium supplemented with the appropriate antibiotic and either arabinose only (to induce expression of Cage and GFP1-10) or both arabinose and IPTG (to induce expression of Cage, GFP1-10, and Key) and grown for 16 hours at 37 °C. 200 μL of each expression culture was washed once with 200 μL PBS, resuspended in 200 μL PBS, and transferred to a black-walled 96-well plate. GFP fluorescence was assessed in a Biotek Synergy H1MF plate reader (excitation / emission 479 / 520 nm). Fluorescence was minimal with Cage alone, confirming that the LOCKR protein prevented activation of split GFP in the absence of Key. Induction of Key expression resulted in a large increase in fluorescence in sequences 27192, 27198, 27194, 27202, 27206, and 27210. These results indicate that the 3 plus 1 LOCKR architecture can control the function of the bioactive peptide GFP11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] All references cited are incorporated herein by reference in their entirety. In this application, unless otherwise noted, techniques utilized may be found in any of several well-known references, such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991 Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (MP Deutscher, ed., (1990) Academic Press, Inc.), PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2 nd (RIFreshney.1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ Murray, The Humana Press Inc., Clifton, NJ), and Ambion 1998 Catalog (Ambion, Austin, TX).

[0023] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, "and" is used interchangeably with "or" unless otherwise noted.

[0024] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0025] All embodiments of any aspect of this disclosure can be used in combination unless the context clearly indicates otherwise.

[0026] Unless the context clearly requires otherwise, throughout the detailed description and claims, the words "comprise," "comprising," and the like, are to be construed in an inclusive or exhaustive sense, i.e., "including, but not limited to," as opposed to an exclusive sense. Words using the singular or plural also include the plural and singular, respectively. Additionally, the words "herein," "above," and "below," and words of similar import, when used in this application, refer to this application as a whole and not to any particular portions of this application.

[0027] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. While specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the art will recognize.

[0028] In a first aspect, the disclosure provides a non-naturally occurring polypeptide, (a) A helical bundle containing 2 to 7 alpha helices; (b) an amino acid linker connecting each alpha helix.

[0029] The non-naturally occurring polypeptides of this first aspect of the disclosure can be used, for example, as the "cage" polypeptide component (also referred to herein as "lock") of the novel protein switches disclosed in detail herein. The protein switches can be used, for example, to sequester bioactive peptides in a cage polypeptide and hold them in an inactive ("off") state until combined with a second component (a "key" polypeptide) of the novel protein switches disclosed herein, which induces a conformational change that activates (turns "on") the bioactive peptide (see FIG. 1A). The polypeptides described herein include the first de novo designed polypeptides that can undergo conformational switching in response to protein binding. Moreover, there are no known naturally occurring proteins that can switch in a modular, tunable manner like the polypeptides disclosed herein.

[0030] A polypeptide is "non-naturally occurring" in that the entire polypeptide is not found in any naturally occurring polypeptide. It will be understood that components of a polypeptide may be naturally occurring, including, but not limited to, biologically active peptides, which may be included in some embodiments.

[0031] The cage polypeptide comprises a helix bundle comprising 2-7 alpha helices, in various embodiments the helix bundle comprises 3-7, 4-7, 5-7, 6-7, 2-6, 3-6, 4-6, 5-6, 2-5, 3-5, 4-5, 2-4, 3-4, 2-3, 2, 3, 4, 5, 6, or 7 alpha helices.

[0032] The design of the helix bundle cage polypeptide of the present disclosure may be carried out by any suitable means. In one non-limiting embodiment, the BundleGridSampler™ of the Rosetta™ program is used to create a backbone shape based on the Crick formula of the coiled coil, allowing efficient and parallel sampling of a regular grid of coiled coil formula parameter values, corresponding to a continuum of peptide backbone conformations. This may be supplemented by designing for hydrogen bond networks using any suitable means, such as but not limited to those reported by Boyken et al. (Science 352, 680-687 (2016)), followed by Rosetta™ side chain design. In a further non-limiting embodiment, the best scoring design based on total score, number of unsatisfactory hydrogen bonds, and lack of voids in the core of the protein may be selected for the helix bundle polypeptide design.

[0033] Each alpha helix can be of any suitable length and amino acid composition suitable for the intended use, hi one embodiment, each helix is ​​independently 38-58 amino acids in length. In various embodiments, each helix is ​​independently 18-60, 18-55, 18-50, 18-45, 22-60, 22-55, 22-50, 22-45, 25-60, 25-55, 25-50, 25-45, 28-60, 28-55, 28-50, 28-45, 32-60, 32-55, 32-50, 32-45, 35-60, 35-55, 35-50, 35-45, 38-60, 38-55, 38-50, 38-45, 40-60, 40-58, 40-55, 40-50, or 40-45 amino acids in length.

[0034] The amino acid linkers connecting each alpha helix can be of any suitable length or amino acid composition appropriate for the intended use, In one non-limiting embodiment, each amino acid linker is independently 2-10 amino acids in length and does not include any additional functional sequences that may be fused to the linker. In various non-limiting embodiments, each amino acid linker is independently 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 2-9, 3-9, 4-9, 5-9, 6-9, 7-9, 8-9, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 2-7, 3-7, 4-7, 5-7, 6-7, 2-6, 3-6, 4-6, 5-6, 2-5, 3-5, 4-5, 2-4, 3-4, 2-3, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long. In all embodiments, the linker can be structured or flexible (e.g., polyGS). These linkers can code for additional functional sequences, including but not limited to, a protease cleavage site or half of a split intein system (see sequences below).

[0035] The polypeptide of this first embodiment comprises a region called the "latch region" for insertion of a bioactive peptide. Thus, the cage polypeptide comprises a latch region and a structural region (i.e., the remainder of the cage polypeptide that is not the latch region). When the latch region is altered to include one or more bioactive peptides, the structural region of the cage polypeptide interacts with the latch region to prevent activity of the bioactive peptides. Upon activation by the key polypeptide, the latch region dissociates from its interaction with the structural region to expose the bioactive peptides and allow the peptides to function.

[0036] As used herein, a "bioactive peptide" is any peptide of any length or amino acid composition that can selectively bind to a defined target (i.e., can bind to an "effector" polypeptide). Such bioactive peptides can include peptides with all three types of secondary structures in the inactive conformation: alpha helices, beta strands, and loops. Polypeptides of this embodiment can be used to control the activity of a wide range of functional peptides. The ability to harness these biological functions with strict, inducible control is useful, for example, in engineering cells (such as inducible activation of functions, engineering complex logic operations and circuits), developing sensors, developing inducible protein-based therapeutics, and creating novel biological materials.

[0037] The latch region may be located near either end of the cage polypeptide. In one embodiment, the latch region is located in the C-terminal helix, thereby positioning the bioactive peptide at the maximum burial of functional residues that need to be sequestered to maintain the bioactive peptide in an inactive state, while simultaneously burying hydrophobic residues to facilitate solvent exposure / compensatory hydrogen bonding of polar residues. In various embodiments, the latch region may comprise part or all of a single alpha helix in the cage polypeptide, at the N- or C-terminal portion. In various other embodiments, the latch region may comprise part or all of the first, second, third, fourth, fifth, sixth, or seventh alpha helix in the cage polypeptide. In other embodiments, the latch region may comprise all or part of two or more different alpha helices in the cage polypeptide, e.g., the C-terminal portion of one alpha helix and the N-terminal portion of the next alpha helix, all two consecutive alpha helices, etc.

[0038] In another embodiment, the polypeptide of this first aspect comprises one or more bioactive peptides in the latch region. In this embodiment, the bioactive peptide(s) may replace one or more amino acids in the latch region or may be added to the latch region without removing any amino acid residues from the latch region. In various non-limiting embodiments, the bioactive peptide may comprise one or more of SEQ ID NOs: 60, 62-64, 66, 27052-27093, and 27118-27119, or variants thereof (all sequences in brackets are optional). GFP11 fluorescent peptide and peptide that binds to GFP1-10: RDHMVLHEYVNAAGIT (sequence number 27052) - BIM binding peptide and apoptotic peptide against BCL-2: IxxxLRxIGDxFxxxY (SEQ ID NO: 50), where x is any amino acid, in one embodiment the peptide is EIWIAQELRRIGDEFNAYYA (SEQ ID NO: 60). Peptide designed to bind to BCL-2: KMAQELIDKVRAASLQINGDAFYAILRAL (SEQ ID NO: 62) StreptagII binding peptide or antibody against streptactin: (N)WSHPQFEK (SEQ ID NO: 63) - TEV protease cleavage site: ENLYFQ(G)-X (SEQ ID NO: 64), where (G) can be S and the last position, X, can be anything except proline. Thrombin protease cleavage site: LVPRGS (SEQ ID NO: 66) Cathepsin cleavage site: RLVGFE (sequence number 27053) Spytag covalent cross-linking peptide with Spycatcher: AHIVMVDAYK (PTK) (SEQ ID NO: 27054) NLS peptide that targets proteins to the nucleus: AAAKRARTS (sequence number 27055) NES1 peptide that excludes proteins from the nucleus: LALKLAGLDIN (SEQ ID NO: 27056) NES2 peptide that excludes proteins from the nucleus: ELAEKLAGLDIN (SEQ ID NO: 27057) NES3 peptide that excludes proteins from the nucleus: ELAEKLRAGLDLN (SEQ ID NO: 27058) EZH2-binding peptide that recruits DNA-methylase: TMFSSNRQKILERTETLNQEWKQRRIQ (SEQ ID NO: 27059) MDM2 binding peptide that recruits p53: ETFSDLWKLL (SEQ ID NO: 27060) CP5 binding peptide: GELDELVYLLDGPGYDPIHSDVVTRGGSHLFNF (sequence number 27061) 9aaTAD1 for transcriptional activation: TMDDVYNYLFDD (sequence number 27062) 9aaTAD2 for transcriptional activation: LLTGLFVQYLFDD (SEQ ID NO: 27063) 9aaTAD3 for transcriptional activation: DDAVVESFFSS (sequence number 27064) 9aaTAD4 for transcriptional activation: GDFLSDLFD (SEQ ID NO: 27065) 9aaTAD5 for transcriptional activation: GDVLSDLVD (sequence number 27066) ●Mad1-SID-Epigenetic modification: NIQMLLEAADYLE (sequence number 27067) ●Mad1-SID (3A mutant)-epigenetic modification: NIAMLLAAAAYLE (sequence number 27068) RHIM domain 1 from ZBP1: IQIG (sequence number 27069) RHIM domain 2 from ZBP1: VQLG (SEQ ID NO: 27070) nanoBit split luciferase: VSGWRLFKKIS (sequence number 27071) ● CC-A: GLEQEIAALEKENAALEWEIAALEQGG (sequence number 27072) ● CC-B: GLKQKIAALKYKNAALKKKIAALKQGG (sequence number 27073) ●GCN4:RMKQLEDKVEELLSKNYHLENEVARLKKLVGER (sequence number 27074) ● CC-Di:GEIAALKQEIAALKKENAALKWEIAALKQG (sequence number 27075) Membrane disrupting / cell penetrating peptides: GALA for membrane disruption: WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 27076) ● Olein 1.2: GLFDIIKKIAESF (sequence number 27077) Magainin-1: GIGKFLHSAGKFGKAFVGEIMKS (sequence number 27078) Magainin-2: GIGKFLHSAKKFGKAFVGEIMNS (sequence number 27079) Melittin: GIGAVLKVLTTGLPALISWIKRKRQQ (sequence number 27080) Mastoparan X: INWKGIAAMAKKLL (sequence number 27081) Cecropin A: KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK (sequence number 27082) Cecropin P1: SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (sequence number 27083) Citropin 1.1: GLFDVIKKVASVIGGL (sequence number 27084) Temporin-1Lb: NFLGTLINLAKKIL (sequence number 27085) HPV33L2 peptide: SYFILRRRRKRFPYFFTDVRVAA (sequence number 27086) Adenovirus pVI membrane fusion domain: AFSWGSLWSGIKNFGSTVKNY (SEQ ID NO: 27087) Gamma-1 peptide from Flock House virus: ASMWERVKSIIKSSLAAASNI (SEQ ID NO: 27088) Poliovirus 2B pore-forming peptide: VTSTITEKLLKNLIKIISSLVIITRNYEDTTTVLATLALLGCDASPWQWL (SEQ ID NO: 27089) Rhinovirus pore-forming peptide: IAQNPVENYIDEVLNEVLVVPNIN (SEQ ID NO: 27090) Influenza HA2 pore-forming peptide: FLGIAEAIDIGNGWEGMEFG (sequence number 27091) Influenza HA2 derivative: GLFGAIAGFIENGWEGMIDG (sequence number 27092) HA-derived INF6: GLFGAIAGFIENGWEGMIDGWYG. (SEQ ID NO: 27093) KRAB domain-epigenetic modification: (SEQ ID NO: 27118) MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLV Minimal Krab domain (KOX111-55)-Epigenetic modification: (SEQ ID NO: 27119) RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY

[0039] In one embodiment, the dynamic range of activation by a key polypeptide can be adjusted by truncating the length of the latch region to be shorter than the alpha helices of the structural region, while simultaneously weakening the cage polypeptide-latch region interaction and opening an exposed region to the cage polypeptide to which the key polypeptide can bind as a "toehold" (Figure 2). Similarly, the dynamic range of activation by a key polypeptide can be adjusted in a similar manner by designing mutations to the latch that weaken the cage polypeptide-latch region interaction (Figures 1-2, and 10). In other embodiments, the latch region can be one or more helices of length between 18-150 amino acids, 18-100 amino acids, 18-58 amino acids, or any range encompassed by these ranges. In other embodiments, the latch region can be a helix secondary structure, a beta strand secondary structure, a loop secondary structure, or a combination thereof.

[0040] In a second aspect, the present disclosure provides SEQ ID NOs: 1-49, 51-52, 54-59, 61, 65, 67-91, 92-2033 (U.S. Provisional Patent Application No. 62 / 700681, filed July 19, 2018, and / or No. 62 / 785537, filed December 27, 2018, which are filed as Exhibit 1), SEQ ID NOs: 2034-14317 (U.S. Provisional Patent Application No. 62 / 700681, filed July 19, 2018, and / or No. 62 / 785537, filed December 27, 2018, which are filed as Exhibit 2), 27094-27117, 27120-27125, 27,278-27,321, and Table 2 (sequences The non-naturally occurring polypeptides provided herein include polypeptides having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along their length to the amino acid sequence of a caged polypeptide disclosed herein, such as a caged polypeptide listed in Table 3, Table 4, Table 5, or Table 6, including any of the caged polypeptides having an even-numbered SEQ ID NO: in the range 27126-27276, Table 3, and / or Table 4. In each embodiment, the N-terminal and / or C-terminal 60 amino acids of each caged polypeptide may be optional, and the terminal 60 amino acid residues may be modified, such as by replacing all or a portion of the latch with a biologically active peptide. In one embodiment, the N-terminal 60 amino acid residues are optional, in another embodiment, the C-terminal 60 amino acid residues are optional, and in a further embodiment, each of the N-terminal 60 amino acid residues and the C-terminal 60 amino acid residues are optional. In one embodiment, these optional N-terminal and / or C-terminal 60 residues are not included in determining the percent sequence identity. In another embodiment, the optional residues may be included in determining the percent sequence identity.

[0041] As disclosed herein, the bioactive peptides sequestered by the polypeptides of the present disclosure are located within the latch region. The latch region is indicated by brackets in the sequence of each cage polypeptide. The bioactive peptides can be added to the latch region without removing any residues of the latch region, or can replace one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids in the cage scaffold latch region to generate the final polypeptide. Thus, the latch region can be significantly modified with the inclusion of the bioactive peptide. In one embodiment, the optional residues are not included in the determination of percent sequence identity. In another embodiment, the latch region residues can be included in determining percent sequence identity. In a further embodiment, the optional residues and each of the latch residues can be not included in determining percent sequence identity.

[0042] In one embodiment of this second aspect, the polypeptide is a polypeptide according to any embodiment or combination of embodiments of the first aspect and has the required 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along its length to the amino acid sequence of a reference cage polypeptide listed herein. In another embodiment, the polypeptide further comprises a bioactive peptide within (or substituting for) the latch region of the cage polypeptide.

[0043] The cage polypeptide may be a cage scaffold (i.e., without a bioactive peptide), e.g., see SEQ ID NOs: 1-17, 2034-14317, and the specific cage polypeptides listed in Table 2, Table 3, and / or Table 4, or may further comprise a bioactive peptide sequestered in the latch region of the cage scaffold polypeptide (present as a fusion with the cage scaffold polypeptide), as described in more detail herein (e.g., see SEQ ID NOs: 18-49, 51-52, 54-59, 61, 65, 67-2033, 27094-27117, 27120-27125, and the specific cage polypeptides listed in Tables 2, 3, and / or 4). In certain embodiments, the caged polypeptide shares 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along their length with the amino acid sequences of the caged polypeptides in Table 2, Table 3, and / or Table 4.

[0044] In another embodiment, the cage polypeptide shares 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along their length with the amino acid sequences of the cage polypeptides in Table 3. In another embodiment, the cage polypeptide shares 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along their length with the amino acid sequences of the cage polypeptides in Table 4. In one embodiment of each of these embodiments, these optional N-terminal and / or C-terminal 60 residues are not included in determining percent sequence identity, hi another embodiment, the optional residues may be included in determining percent sequence identity.

[0045] As disclosed in the Examples below, exemplary cage and key polypeptides of the present disclosure have been identified and mutational analysis has been performed. Moreover, different designs starting from the same exemplary cage and key polypeptides result in different amino acid sequences while maintaining the same intended function. In various embodiments, a given amino acid can be substituted with a residue having similar physicochemical properties, for example, one aliphatic residue can be substituted for another (such as Ile, Val, Leu, or Ala for each other), or one polar residue can be substituted for another (such as between Lys and Arg, Glu and Asp, or Gln and Asn). Other such conservative substitutions are known, for example, substitutions of entire regions with similar hydrophobic properties. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity is retained. Amino acids can be classified according to similarities in the properties of their side chains (ALLehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)). (1) Non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M), (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q), (3) Acidic: Asp (D), Glu (E), (4) Basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be classified based on common side chain properties. (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) acidic: Asp, Glu, (4) basic: His, Lys, Arg, (5) residues that affect chain orientation: Gly, Pro, (6) aromatic: Trp, Tyr, Phe. Nonconservative substitutions involve exchanging a member of one of these classes for another class.Particular conservative substitutions include, for example, Ala to Gly or Ser, Arg to Lys, Asn to Gln or His, Asp to Glu, Cys to Ser, Gln to Asn, Glu to Asp, Gly to Ala or Pro, His to Asn or Gln, Ile to Leu or Val, Leu to Ile or Val, Lys to Arg, Gln, or Glu, Met to Leu, Tyr, or Ile, Phe to Met, Leu, or Tyr, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr to Trp, and / or Phe to Val, Ile, or Leu.

[0046] Exemplary caged polypeptides (see also SEQ ID NOs: 92-14317, 27094-27117, 27120-27125, 27,728-27321, and the caged polypeptides listed in Tables 2, 3, and / or 4): 1) Exemplary reference cage polypeptides: Latch regions are indicated in brackets [ ] ●6His-MBP-TEV, 6His-TEV, and flexible linker sequences are underlined. The fused functional domains (DARPin, split intein components, and fluorescent protein) are shown in bold. ●Functional peptides are shown in italics and underlined. Exemplary positions that have been mutated to any amino acid to modulate responsiveness are shown in bold and underlined. These positions are exemplary and are not a complete list of residues that can modulate responsiveness. C-terminal sequences that can be deleted to modulate responsiveness are included in parentheses. A range from position one (1) to all residues included in the parentheses can be removed, starting at the C-terminus and deleting consecutive residues therein. All sequences within brackets are optional >SB76L (SEQ ID NO: 1) [ka] >SB76L_17 (SEQ ID NO:2) [ka] >SB76L_18 (SEQ ID NO: 3) [ka] >LOCKR_extend5(sequence number 4) [ka] >LOCKR_extend9 (sequence number 5) [ka] >LOCKR_extend18 (sequence number 6) [ka] >LOCKRb (SEQ ID NO: 7) [ka] >LOCKRc (sequence number 8) [ka] >LOCKRd (SEQ ID NO: 9) [ka] >LOCKRe (sequence number 10) [ka] >LOCKRf (SEQ ID NO: 11) [ka] >miniLOCKRa_1 (SEQ ID NO: 12) [ka] >miniLOCKRa_2 (SEQ ID NO: 13) [ka] >miniLOCKRc_1 (SEQ ID NO: 14) [ka] >miniLOCKRc_2 (sequence number 15) [ka] >1fix-short-noBim-t0 (sequence number 16) [ka] >1fix-short-noBim(AYYA)-t0 (SEQ ID NO: 17) [ka] (3) Design of functional LOCKR cages with bioactive peptides encoded in the latch. >aBcl2LOCKR (SEQ ID NO: 18) [ka] >pBimLOCKR (SEQ ID NO: 19) [ka] >BimLOCKR_extend5 (sequence number 20) [ka] >BimLOCKR_extend9 (sequence number 21) [ka] >BimLOCKR_extend18 (sequence number 22) [ka] >BimLOCKRb (SEQ ID NO: 23) [ka] >BimLOCKRc (SEQ ID NO: 24) [ka] >BimLOCKRd (SEQ ID NO: 25) [ka] >StrepLOCKRa_300 (SEQ ID NO: 26) [ka] >strepLOCKRa_306 (SEQ ID NO: 27) [ka] >strepLOCKRa_309 (SEQ ID NO: 28) [ka] >strepLOCKRa_312 (SEQ ID NO: 29) [ka] >strepLOCKRa_313 (SEQ ID NO: 30) [ka] >strepLOCKRa_317 (SEQ ID NO: 31) [ka] >strepLOCKRa_320 (SEQ ID NO: 32) [ka] >strepLOCKRa_323 (SEQ ID NO: 33) [ka] >strepLOCKRa_329 (SEQ ID NO: 34) [ka] >SB13_LOCKR (SEQ ID NO: 35) [ka] >ZCX12_LOCKR (sequence number 36) [ka] >SB13_LOCKR_extend18 (SEQ ID NO: 37) [ka] >ZCX12_LOCKR_extend18 (sequence number 38) [ka] >fretLOCKRa (SEQ ID NO: 39) [ka] >fretLOCKRb (SEQ ID NO: 40) [ka] >fretLOCKRc (sequence number 41) [ka] >fretLOCKRd (sequence number 42) [ka] >tevLOCKR (SEQ ID NO:43) [ka] >spyLOCKR (sequence number 44) [ka] >1_nesLOCKR (sequence number 45) [ka] >2_nesLOCKR (sequence number 46) [ka] >3_nesLOCKR (sequence number 47) [ka] >nlsLOCKR (sequence number 48) [ka] >ezh2LOCKR (SEQ ID NO:49) [ka] >1fix_VMAc_C_BIMlatcht9 (sequence number 51) [ka] >sfGFP_VMAn_1fix_BIM_t0_latch (sequence number 52) [ka] Asymmetric functional cages encoding Bim and GFP11 (i.e., bioactive peptides) (6His-MBP-TEV, 6His-TEV, and flexible linker sequences are underlined.) (Colocalization domains are shown in bold.) (Functional peptides are underlined and italicized.) (Positions that can be mutated to any amino acid to modulate responsiveness are shown in bold and underlined.) (C-terminal sequences that can be deleted to modulate responsiveness are shown in italics.) (All sequences within brackets are optional) >1fix-long-BIM-t0 (SEQ ID NO:54) [ka] >1fix-long-GFP-t0 (SEQ ID NO:55) [ka] >1fix-short-BIM-t0 (sequence number 56) [ka] >1fix-short-GFP-t0 (SEQ ID NO:57) [ka] >Spycatcher-1fix-long-GFP-t0 (SEQ ID NO:58) [ka] >Spycatcher-1fix-short-GFP-t0 (SEQ ID NO:59) [ka] >1fix-latch_Mad1SID_t0_1 (sequence number 61) [ka] >1fix-latch_Mad1SID_T0_2 (sequence number 65) [ka] >1fix-short-Bim-t0-relooped (sequence number 67) [ka] >1fix-short-spytag-t0_2 (sequence number 68) [ka] >1fix-short-spytag-t0_8 (sequence number 69) [ka] >1fix-short-TEV-t0_1 (sequence number 70) [ka] >1fix-short-TEV-t0_6 (array number 71)

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[0047] In a fourth aspect, the present disclosure provides SEQ ID NOs: 14318-26601 (filed as Exhibit 3 in U.S. Provisional Patent Application No. 62 / 700681 filed July 19, 2018 and / or No. 62 / 785537 filed December 27, 2018), 26602-27015 (filed as Exhibit 3 in U.S. Provisional Patent Application No. 62 / 700681 filed July 19, 2018 and / or No. 62 / 785537 filed December 27, 2018, 3plus1_GFP11_Key_Cterm_1" Nos. 1-67 and 97-117, 3plus1_GFP11_Key_Nterm_1" Nos. 68-96 and 118-140, 2plus1_GFP11_Key_Cterm_" Nos. 1-173, and and GFP11_Key_Nterm_"Nos. 174-274), 27016-27050, 27,322-27,358, and the key polypeptides in Table 2 (polypeptides having odd-numbered SEQ ID NOs. between 27127-27277), Table 3, and / or Table 4, including polypeptides having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along its length, not including any amino acids, to an amino acid sequence of a key polypeptide selected from the key polypeptides in Table 2 (polypeptides having odd-numbered SEQ ID NOs. between 27127-27277), Table 3, and / or Table 4.

[0048] As disclosed herein, a polypeptide of this embodiment can be used, for example, as a key polypeptide coupled to a cage polypeptide to displace the latch by competitive intermolecular binding that induces a conformational change to expose an encoded bioactive peptide or region, thus activating the system (see, e.g., FIG. 1).

[0049] As described in this disclosure, key polypeptides may include residues that are optional, which are indicated in brackets and in one embodiment are not included in determining percent sequence identity, hi another embodiment, optional residues may be included in determining percent sequence identity.

[0050] In another embodiment, the non-naturally occurring polypeptide comprises a polypeptide having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along its length to an amino acid sequence of a key polypeptide selected from the group consisting of SEQ ID NOs: 26602-27050, and 27,322-27,358, and are described in more detail below. ●The key layout is normal characters. ●6His-MBP-TEV, 6His-TEV, and flexible linker sequences are underlined. - The bold and italicized sequences are optional residues required for biotinylation of MBP_key. All sequences within brackets are optional - Any number of consecutive amino acids at the N- or C-terminus of the non-optional key sequence may be deleted to adjust responsiveness. >SB76_C-helix (sequence number 27016) DEARKAIARVKRESKRIVEDAERLIREAAAASEKIS >SB76_C-helix-biotin (SEQ ID NO: 27017) DEARKAIARVKRESKRIVEDAERLIREAAAASEKISGSGK-Biotin >p5_MBP (sequence number 27018) [ka] >p9_MBP (sequence number 27019) [ka] >p18_MBP (sequence number 27020) [ka] >MBP_p18 (alias p76) (SEQ ID NO: 27021) [ka] >key_b (sequence number 27022) [ka] >key_c (sequence number 27023) [ka] >key_d(sequence number 27024) [ka] >key_e(sequence number 27025) [ka] >key_f(sequence number 27026) [ka] Additional keys: The keyboard layout is normal characters (6His-MBP-TEV, 6His-TEV, and flexible linker sequences are underlined.) (Colocalization domains are shown in bold.) (Positions that can be mutated to any amino acid to modulate responsiveness are shown in bold and underlined. These are exemplary and not exhaustive.) (Any number of consecutive amino acids at the N- or C-terminus of the non-optional key sequence may be deleted to adjust responsiveness.) (All sequences within brackets are optional) >p76-long (sequence number 27027) [ka] >p76-short (sequence number 27028) [ka] >k76-long (sequence number 27029) [ka] >k76-short (sequence number 27030) [ka] >p76_GLISE (sequence number 27031) [ka] >p76_GSSEKIS (sequence number 27032) [ka] >p76_R26G (sequence number 27033) [ka] >p76-short_E19G (sequence number 27034) [ka] >p76-short_GLISE_E01_EGFR (sequence number 27035) [ka] >p76-short_AE_EGFR (SEQ ID NO: 27036) [ka] >p76-short_AAE_EGFR (SEQ ID NO: 27037) [ka] >p76-short_EE_EGFR (sequence number 27038) [ka] >p76-Spitag (SEQ ID NO: 27039) [ka] >p76-short-spytag (sequence number 27040) [ka] >sfGFP_VMAn_p18 (sequence number 27041) [ka] >p18_VMAc_mCherry (sequence number 27042) [ka] (2plus1 and 3plus1STREPII-LOCK® functional cage design with homologous keys): >2plus1_KEY_100000.fastaalt_STREP_2plus1_1 (sequence number 27043) DKVRKVAEVAEKVLRDIDKLDRESKEAFRRTNGEISKLDEDTRRVAERVKKAIEDLAK >2plus1_KEY_2 (sequence number 27044) SEVDEIIADNERALDEVRREVEEIDKENAERLKEWVEEAREILDRLAKALEEIR >2plus1_KEY_3 (sequence number 27045) PEEALSKAIKDVRDIVKKVKDELKEWRDRNKELVDRLSEELKEWLKDVERVLKELTDKDR >2plus1_KEY_4 (sequence number 27046) DERVREELKKLLTRVEEEHRKVLETDKKILKEAHKESKEVNDRDRELLERLEESVR >3plus1_KEY_1 (sequence number 27047) SRLVKKLDEIVKEVAKKLEDVVRANEELWRKLVELNKESVARLREAVERVARDLEETAR >3plus1_KEY_2 (sequence number 27048) SDEERLEKVVKDVIEKVRRILEKWKKDIDKVVKELRRILEEWEKIIREVLDKVR >3plus1_KEY_3 (sequence number 27049) DKDAVIKVIEKLIRANAAVWDALLKINEDLVRVNKTVWKELLRVNEKLARDLERVVK >3plus1_KEY_4 (sequence number 27050) SLVDELRKSLERNVRVSEEVARRLKEALKRWVDVVRKVVEDLIRLNEDVVRVVEK SEQ ID NOs: 26,602 to 27,015: >3plus1_GFP11_Key_Cterm_1 (sequence number 26602) SGSKEVLDILERAVEVVRRVIKALKEVLERHVDATREVIERVKRVNKRLLEAVREVVT >3plus1_GFP11_Key_Cterm_2 (sequence number 26603) GVPEEIDRELKRVVEELRRLHEEIKERLDDVARRSEEELRRIIKKLKEVVKEIRKKLK >3plus1_GFP11_Key_Cterm_3 (sequence number 26604) DLLRKLEEELRRIKEKLRKALEELEREHRELEKELDKLHDESRKEHERIEEELRR >3plus1_GFP11_Key_Cterm_4 (sequence number 26605) DEDLLEKIKRVIREHIKALEKLARDLKEILRRHIEALKELARDLAEVIRKLLEDVKR >3plus1_GFP11_Key_Cterm_5 (sequence number 26606) DLERLRRKVEELEDRRRLLLEKLARDSAELMRELERILDRYARESEELDRRLAE >3plus1_GFP11_Key_Cterm_6 (sequence number 26607) DLEDILRKNLDRLRKLLERLREILRENLEALKKTLKRLEDVVREILEDLKRERK >3plus1_GFP11_Key_Cterm_7 (sequence number 26608) DLERLRRKVEELEDRRRLLLEKLARDSAELMRELERILDRYARESEELDRRLAE >3plus1_GFP11_Key_Cterm_8 (sequence number 26609) SGSKEVLDILERAVEVVRRVIKALKEVLERHVDATREVIERVKRVNKRLLEAVREVVT >3plus1_GFP11_Key_Cterm_9 (sequence number 26610) DLERLRRKVEELEDRRRLLLEKLARDSAELMRELERILDRYARESEELDRRLAE >3plus1_GFP11_Key_Cterm_10 (sequence number 26611) RLIEEVVRLLRENLDVVRRILEALAKLIKELLEALEEVLRRNKELIRELLRVLDEALK >3plus1_GFP11_Key_Cterm_11 (sequence number 26612) DIVRAMEEVIRRLIEILRRDVELNLDVAKKLLELLKEDSKLNLDVARELLELLDR >3plus1_GFP11_Key_Cterm_12 (sequence number 26613) DIVRAMEEVIRRLIEILRRDVELNLDVAKKLLELLKEDSKLNLDVARELLELLDR >3plus1_GFP11_Key_Cterm_13 (sequence number 26614) RLIEEVVRLLRENLDVVRRILEALAKLIKELLEALEEVLRRNKELIRELLRVLDEALK >3plus1_GFP11_Key_Cterm_14 (sequence number 26615) RLIEEVVRLLRENLDVVRRILEALAKLIKELLEALEEVLRRNKELIRELLRVLDEALK >3plus1_GFP11_Key_Cterm_15 (sequence number 26616) DLLRKLEEELRRIKEKLRKALEELEREHRELEKELDKLHDESRKEHERIEEELRR >3plus1_GFP11_Key_Cterm_16 (sequence number 26617) DLLRKLEEELRRIKEKLRKALEELEREHRELEKELDKLHDESRKEHERIEEELRR >3plus1_GFP11_Key_Cterm_17 (sequence number 26618) ELAREVERVIKELLDKSKEILERIERAIDELLKVSEEILKLSEDASEELLKILREFAK >3plus1_GFP11_Key_Cterm_18 (sequence number 26619) DVKDIIRTILEVARDLLRLLEEDSRTNSEVVKRLLDLLREDSKANSEVVKRLLDVLRE >3plus1_GFP11_Key_Cterm_19 (sequence number 26620) DLERLRRKVEELEDRRRLLLEKLARDSAELMRELERILDRYARESEELDRRLAE >3plus1_GFP11_Key_Cterm_20 (sequence number 26621) DLERLRRKVEELEDRRRLLLEKLARDSAELMRELERILDRYARESEELDRRLAE >3plus1_GFP11_Key_Cterm_21 (sequence number 26622) RLIEEVVRLLRENLDVVRRILEALAKLIKELLEALEEVLRRNKELIRELLRVLDEALK >3plus1_GFP11_Key_Cterm_22 (sequence number 26623) DLEDILRKNLDRLRKLLERLREILRENLEALKKTLKRLEDVVREILEDLKRERK >3plus1_GFP11_Key_Cterm_23 (sequence number 26624) DLLRKLEEELRRIKEKLRKALEELEREHRELEKELDKLHDESRKEHERIEEELRR >3plus1_GFP11_Key_Cterm_24 (sequence number 26625) DEDLLEKIKRVIREHIKALEKLARDLKEILRRHIEALKELARDLAEVIRKLLEDVKR >3plus1_GFP11_Key_Cterm_25 (sequence number 26626) ELVRIAIEVLKRLLEIIEELVRLNNEILERLLKIVRELHKDNIKILEDLLRIIEEVLR >3plus1_GFP11_Key_Cterm_26 (sequence number 26627) ELVRIAIEVLKRLLEIIEELVRLNNEILERLLKIVRELHKDNIKILEDLLRIIEEVLR >3plus1_GFP11_Key_Cterm_27 (sequence number 26628) RLARLLKALADKLIRVLEEILKINEELNRKIIKFARENLERNRRVNKKVIEVLREAAR >3plus1_GFP11_Key_Cterm_28 (sequence number 26628) DLERLRRKVEELEDRRRLLLEKLARDSAELMRELERILDRYARESEELDRRLAE >3plus1_GFP11_Key_Cterm_29 (sequence number 26630) ELVRIAIEVLKRLLEIIEELVRLNNEILERLLKIVRELHKDNIKILEDLLRIIEEVLR >3plus1_GFP11_Key_Cterm_30 (sequence number 26631) ELVRIAIEVLKRLLEIIEELVRLNNEILERLLKIVRELHKDNIKILEDLLRIIEEVLR >3plus1_GFP11_Key_Cterm_31 (sequence number 26632) DIVRAMEEVIRRLIEILRRDVELNLDVAKKLLELLKEDSKLNLDVARELLELLDR >3plus1_GFP11_Key_Cterm_32 (sequence number 26633) RKIAKIIEELKRLLEDLARDTRRVIEEAKRLLKEWRDRNKEVADTLKKLLEDLIRKIR >3plus1_GFP11_Key_Cterm_33 (sequence number 26634) DLLRKLEEELRRIKEKLRKALEELEREHRELEKELDKLHDESRKEHERIEEELRR >3plus1_GFP11_Key_Cterm_34 (sequence number 26635) DLLRKLEEELRRIKEKLRKALEELEREHRELEKELDKLHDESRKEHERIEEELRR >3plus1_GFP11_Key_Cterm_35 (sequence number 26636) RKIAKIIEELKRLLEDLARDTRRVIEEAKRLLKEWRDRNKEVADTLKKLLEDLIRKIR >3plus1_GFP11_Key_Cterm_36 (sequence number 26637) ELVRIAIEVLKRLLEIIEELVRLNNEILERLLKIVRELHKDNIKILEDLLRIIEEVLR >3plus1_GFP11_Key_Cterm_37 (sequence number 26638) TVRRLREALKKLEDDLRKIERDAEREYKKLKDELEELTERYRREIRKLKEEELKADRK >3plus1_GFP11_Key_Cterm_38 (sequence number 26639) DEAERRRRELKDKLDRLREEHEEVKRRLEEELTRLRETHKKIEKELREALKRVRDRST >3plus1_GFP11_Key_Cterm_39 (sequence number 26640) DEAERRRRELKDKLDRLREEHEEVKRRLEEELTRLRETHKKIEKELREALKRVRDRST >3plus1_GFP11_Key_Cterm_40 (sequence number 26641) DLEDILRKNLDRLRKLLERLREILRENLEALKKTLKRLEDVVREILEDLKRERK >3plus1_GFP11_Key_Cterm_41 (sequence number 26642) DLERLRRKVEELEDRRRLLLEKLARDSAELMRELERILDRYARESEELDRRLAE >3plus1_GFP11_Key_Cterm_42 (sequence number 26643) DEAERRRRELKDKLDRLREEHEEVKRRLEEELTRLRETHKKIEKELREALKRVRDRST >3plus1_GFP11_Key_Cterm_43 (sequence number 26644) DEAERRRRELKDKLDRLREEHEEVKRRLEEELTRLRETHKKIEKELREALKRVRDRST >3plus1_GFP11_Key_Cterm_44 (sequence number 26645) DEAERRRRELKDKLDRLREEHEEVKRRLEEELTRLRETHKKIEKELREALKRVRDRST >3plus1_GFP11_Key_Cterm_45 (sequence number 26646) DKAVEELEKALEEIKRRLKEVIDRYEDELRKLRKEYKEKIDKYERKLEEIERRERT >3plus1_GFP11_Key_Cterm_46 (sequence number 26647) DVKRALEELVSRLRKLLEDVKKASEDIVREVERIVRELAKRSDEILKKLEDIVEKLRE >3plus1_GFP11_Key_Cterm_47 (sequence number 26648) DVKRALEELVSRLRKLLEDVKKASEDIVREVERIVRELAKRSDEILKKLEDIVEKLRE >3plus1_GFP11_Key_Cterm_48 (sequence number 26649) DEAERRRRELKDKLDRLREEHEEVKRRLEEELTRLRETHKKIEKELREALKRVRDRST >3plus1_GFP11_Key_Cterm_49 (sequence number 26650) DVKRALEELVSRLRKLLEDVKKASEDIVREVERIVRELAKRSDEILKKLEDIVEKLRE >3plus1_GFP11_Key_Cterm_50 (sequence number 26651) EVKRRLEEKERRIRTRYEELRRRLRKRVKDYEDKLREIEKKVRRDAERIEEELERAKK >3plus1_GFP11_Key_Cterm_51 (sequence number 26652) DEAERRRRELKDKLDRLREEHEEVKRRLEEELTRLRETHKKIEKELREALKRVRDRST >3plus1_GFP11_Key_Cterm_52 (sequence number 26653) KIAEEIERELEELRRMIKRLHEDLERKLKESEDELREIEARLEEKIRRLEEKLERKRR >3plus1_GFP11_Key_Cterm_53 (sequence number 26654) KIAEEIERELEELRRMIKRLHEDLERKLKESEDELREIEARLEEKIRRLEEKLERKRR >3plus1_GFP11_Key_Cterm_54 (sequence number 26655) DKAVEELEKALEEIKRRLKEVIDRYEDELRKLRKEYKEKIDKYERKLEEIERRERT >3plus1_GFP11_Key_Cterm_55 (sequence number 26656) KIAEEIERELEELRRMIKRLHEDLERKLKESEDELREIEARLEEKIRRLEEKLERKRR >3plus1_GFP11_Key_Cterm_56 (sequence number 26657) ELVRIAIEVLKRLLEIIEELVRLNNEILERLLKIVRELHKDNIKILEDLLRIIEEVLR >3plus1_GFP11_Key_Cterm_57 (sequence number 26658) DEVEREIRRVKEDLDRILEEYRRLLEEIKRKLEEILRRVEELHRRLRRKLEEIDR >3plus1_GFP11_Key_Cterm_58 (sequence number 26659) DVKRALEELVSRLRKLLEDVKKASEDIVREVERIVRELAKRSDEILKKLEDIVEKLRE >3plus1_GFP11_Key_Cterm_59 (sequence number 26660) DEAERRRRELKDKLDRLREEHEEVKRRLEEELTRLRETHKKIEKELREALKRVRDRST >3plus1_GFP11_Key_Cterm_60 (sequence number 26661) DEAERRRRELKDKLDRLREEHEEVKRRLEEELTRLRETHKKIEKELREALKRVRDRST >3plus1_GFP11_Key_Cterm_61 (sequence number 26662) TLREVVRKVLEEAKRLLDELEEVHKRVKKELEDIIEENRRVVKRVRDELREIKRELDE >3plus1_GFP11_Key_Cterm_62 (sequence number 26663) DVKRALEELVSRLRKLLEDVKKASEDIVREVERIVRELAKRSDEILKKLEDIVEKLRE >3plus1_GFP11_Key_Cterm_63 (sequence number 26664) DVKRALEELVSRLRKLLEDVKKASEDIVREVERIVRELAKRSDEILKKLEDIVEKLRE >3plus1_GFP11_Key_Cterm_64 (sequence number 26665) DEAERRRRELKDKLDRLREEHEEVKRRLEEELTRLRETHKKIEKELREALKRVRDRST >3plus1_GFP11_Key_Cterm_65 (sequence number 26666) DEAERRRRELKDKLDRLREEHEEVKRRLEEELTRLRETHKKIEKELREALKRVRDRST >3plus1_GFP11_Key_Cterm_66 (sequence number 26667) KIAEEIERELEELRRMIKRLHEDLERKLKESEDELREIEARLEEKIRRLEEKLERKRR >3plus1_GFP11_Key_Cterm_67 (sequence number 26668) DVKRALEELVSRLRKLLEDVKKASEDIVREVERIVRELAKRSDEILKKLEDIVEKLRE >3plus1_GFP11_Key_Nterm_68 (sequence number 26669) SEAERLADEVRKAVKKSEEDNETLVREVEKAVRELKKNNKTWVDEVRKLMKRLVDLLR >3plus1_GFP11_Key_Nterm_69 (sequence number 26670) SEAERLADEVRKAVKKSEEDNETLVREVEKAVRELKKNNKTWVDEVRKLMKRLVDLLR >3plus1_GFP11_Key_Nterm_70 (sequence number 26671) DKDKRLEELLKRLKELNDKTFEELERILEELKRANEASLREAERILEELRARIEGGNL >3plus1_GFP11_Key_Nterm_71 (sequence number 26672) SEAEDLEELIKELAELLKDVIRKLEKINRRLVKILEDIIRRLKEISKEAEEELRKGTV >3plus1_GFP11_Key_Nterm_72 (sequence number 26673) SDKEEIKRRVEKTARDLETEHDKIKKRLEDTVRDIKRELDELLEKYERVLRKIEKTLR >3plus1_GFP11_Key_Nterm_73 (sequence number 26674) SEAEKIREALETNLRLLEELIKRLKEILDTHNELLRRVIETLERLLKELLELLEEGGL >3plus1_GFP11_Key_Nterm_74 (sequence number 26675) SEAEKIREALETNLRLLEELIKRLKEILDTHNELLRRVIETLERLLKELLELLEEGGL >3plus1_GFP11_Key_Nterm_75 (sequence number 26676) SKEERLREVAEKHKKDLEDIVKRVDEAAKETARRLEEILKRLEEVLKKILDDLEKGPD >3plus1_GFP11_Key_Nterm_76 (sequence number 26677) SLEEITKRLLELVEENLARHEEILRELLELAKRLAKEDRDILEEVLKLIEELLKLLED >3plus1_GFP11_Key_Nterm_77 (sequence number 26678) SKEETLKRLLDELEKRNRETVERLERLLKELEDRNRASLEELEAVLEELERKIEESGL >3plus1_GFP11_Key_Nterm_78 (sequence number 26679) SKEETLKRLLDELEKRNRETVERLERLLKELEDRNRASLEELEAVLEELERKIEESGL >3plus1_GFP11_Key_Nterm_79 (sequence number 26680) SKEETLKRLLDELEKRNRETVERLERLLKELEDRNRASLEELEAVLEELERKIEESGL >3plus1_GFP11_Key_Nterm_80 (sequence number 26681) STREKAKKVLDTLRADNEDMKRVVEKILRALKRTNERAEKLAREITEEIKRILKEVGV >3plus1_GFP11_Key_Nterm_81 (sequence number 26682) DAEEVVKRLADVLRENDETIRKVVEDLVRIAEENDRLWKKLVEDIAEILRRIVELLRR >3plus1_GFP11_Key_Nterm_82 (sequence number 26683) SKEETLKRLLDELEKRNRETVERLERLLKELEDRNRASLEELEAVLEELERKIEESGL >3plus1_GFP11_Key_Nterm_83 (sequence number 26684) STREKAKKVLDTLRADNEDMKRVVEKILRALKRTNERAEKLAREITEEIKRILKEVGV >3plus1_GFP11_Key_Nterm_84 (sequence number 26685) SKEEEVEKVLRKWEEILRRLIEENKRANDKIRREYEELVKEIRRVLEEIKEVAERLGV >3plus1_GFP11_Key_Nterm_85 (sequence number 26686) DREKSVRDIEEDLKRVLDKLRRRVETSKEELKKVLKADKENADELEKTLRDVVRELDR >3plus1_GFP11_Key_Nterm_86 (sequence number 26687) SDKEEIKRRVEKTARDLETEHDKIKKRLEDTVRDIKRELDELLEKYERVLRKIEKTLR >3plus1_GFP11_Key_Nterm_87 (sequence number 26688) STREKAKKVLDTLRADNEDMKRVVEKILRALKRTNERAEKLAREITEEIKRILKEVGV >3plus1_GFP11_Key_Nterm_88 (sequence number 26689) SKDEELARLLEELVERWRKIVEDLERDHRRLVKEIRELVERIRKKLEELVDRIRKNGI >3plus1_GFP11_Key_Nterm_89 (sequence number 26690) SEAERLADEVRKAVKKSEEDNETLVREVEKAVRELKKNNKTWVDEVRKLMKRLVDLLR >3plus1_GFP11_Key_Nterm_90 (sequence number 26691) SKDEELARLLEELVERWRKIVEDLERDHRRLVKEIRELVERIRKKLEELVDRIRKNGI >3plus1_GFP11_Key_Nterm_91 (sequence number 26692) KEIEETLKELEDLNREMVETNRRVLEETRRLNKETVDRVKATLDELAKMLKKLVDDVR >3plus1_GFP11_Key_Nterm_92 (sequence number 26693) SEAERLADEVRKAVKKSEEDNETLVREVEKAVRELKKNNKTWVDEVRKLMKRLVDLLR >3plus1_GFP11_Key_Nterm_93 (sequence number 26694) SKEETLKRLLDELEKRNRETVERLERLLKELEDRNRASLEELEAVLEELERKIEESGL >3plus1_GFP11_Key_Nterm_94 (sequence number 26695) DKAEVLREALKLLKDLEELIKIHEESLKRILDLIDTLVKVHEDALRALKELLERSGL >3plus1_GFP11_Key_Nterm_95 (sequence number 26696) SKEEEVEKVLRKWEEILRRLIEENKRANDKIRREYEELVKEIRRVLEEIKEVAERLGV >3plus1_GFP11_Key_Nterm_96 (sequence number 26697) SKEETLKRLLDELEKRNRETVERLERLLKELEDRNRASLEELEAVLEELERKIEESGL >3plus1_GFP11_Key_Cterm_97 (sequence number 26698) SERVKEILERILRVVEEAVRLNEESLRRILDVVRKAVKLDRESLKKILDVVEEAVR >3plus1_GFP11_Key_Cterm_98 (sequence number 26699) SERVKEILERILRVVEEAVRLNEESLRRILDVVRKAVKLDRESLKKILDVVEEAVR >3plus1_GFP11_Key_Cterm_99 (sequence number 26700) DERRIAERIRELLRESKKLVRDVVEEAKRLLKENRDSTRKIIEDIRRLLRKIEDSTR >3plus1_GFP11_Key_Cterm_100 (sequence number 26701) DALSRLLEELLRVVDDLIRVLKELIDKSRKVIEELLELLKRINEENLKVLAEIIK >3plus1_GFP11_Key_Cterm_101 (sequence number 26702) DERRIAERIRELLRESKKLVRDVVEEAKRLLKENRDSTRKIIEDIRRLLRKIEDSTR >3plus1_GFP11_Key_Cterm_102 (sequence number 26703) EALRKLVELLVEVLRRLIRVNRELVKLLREVLERLLRILRESVKKLKRLIEKVIKDAT >3plus1_GFP11_Key_Cterm_103 (sequence number 26704) EALRKLVELLVEVLRRLIRVNRELVKLLREVLERLLRILRESVKKLKRLIEKVIKDAT >3plus1_GFP11_Key_Cterm_104 (sequence number 26705) AAKRLVEELLKAVTDLSRKNKRILEELLKAIETLSDENKKILTEILDALRRLVEKIEK >3plus1_GFP11_Key_Cterm_105 (sequence number 26706) EALRKLVELLVEVLRRLIRVNRELVKLLREVLERLLRILRESVKKLKRLIEKVIKDAT >3plus1_GFP11_Key_Cterm_106 (sequence number 26707) EALRKLVELLVEVLRRLIRVNRELVKLLREVLERLLRILRESVKKLKRLIEKVIKDAT >3plus1_GFP11_Key_Cterm_107 (sequence number 26708) DALSRLLEELLRVVDDLIRVLKELIDKSRKVIEELLELLKRINEENLKVLAEIIK >3plus1_GFP11_Key_Cterm_108 (sequence number 26709) DALSRLLEELLRVVDDLIRVLKELIDKSRKVIEELLELLKRINEENLKVLAEIIK >3plus1_GFP11_Key_Cterm_109 (sequence number 26710) RAVKKLDEIVKEVAKKLEDVVRANEELWRALVELNKESVRRLREIVERVARDLEETAR >3plus1_GFP11_Key_Cterm_110 (sequence number 26711) DRLDKVEELVKKLLEDTKRTVDRVRELVRKILKKSRETLEELERLIEKILRELEKDAR >3plus1_GFP11_Key_Cterm_111 (sequence number 26712) DALSRLLEELLRVVDDLIRVLKELIDKSRKVIEELLELLKRINEENLKVLAEIIK >3plus1_GFP11_Key_Cterm_112 (sequence number 26713) RAVKKLDEIVKEVAKKLEDVVRANEELWRALVELNKESVRRLREIVERVARDLEETAR >3plus1_GFP11_Key_Cterm_113 (sequence number 26714) RAVKKLDEIVKEVAKKLEDVVRANEELWRALVELNKESVRRLREIVERVARDLEETAR >3plus1_GFP11_Key_Cterm_114 (sequence number 26715) SEDDLKRVVDEVEKKLRELKRRYAEALERIKEKIKELKDRYERAVREVVAELRKTTK >3plus1_GFP11_Key_Cterm_115 (sequence number 26716) RAVKKLDEIVKEVAKKLEDVVRANEELWRALVELNKESVRRLREIVERVARDLEETAR >3plus1_GFP11_Key_Cterm_116 (sequence number 26717) DEVEREIRRVKEDLDRILEEYRRLLEEIKRKLEEILRRVEELHRRLRRKLEEIDR >3plus1_GFP11_Key_Cterm_117 (sequence number 26718) SEDDLKRVVDEVEKKLRELKRRYAEALERIKEKIKELKDRYERAVREVVAELRKTTK >3plus1_GFP11_Key_Nterm_118 (sequence number 26719) DEAKELLDEIRKAVKESEDRLEKLLRDYEKELRRLEKELRDLKRRIEEKLEELRRGSL >3plus1_GFP11_Key_Nterm_119 (sequence number 26720) SEKEDAARKLRKLVEELTREYEELVKKLERLIEEIEKVSEESVRKLEKLLAEISEEVR >3plus1_GFP11_Key_Nterm_120 (sequence number 26721) SEDEIIKKIIEDLRRVLKEVEEIHKEVEERLDKVLKEAEEMHKEVLKELDRVLDEVKR >3plus1_GFP11_Key_Nterm_121 (sequence number 26722) SKAEEIAEKLDRLLEENRRALEEITTRLDDLLRRNKDALRKVMEKLKRLLDDLRRGGI >3plus1_GFP11_Key_Nterm_122 (sequence number 26723) SEKEKLLKESEEEVRRLRRTLEELLRKYREVLERLRKELREIEERVRDVVRRLKEVLD >3plus1_GFP11_Key_Nterm_123 (sequence number 26724) SEKEDAARKLRKLVEELTREYEELVKKLERLIEEIEKVSEESVRKLEKLLAEISEEVR >3plus1_GFP11_Key_Nterm_124 (sequence number 26725) SKEETLRKEAEDLLRRLEELTRRLEKKARELLERAKKLSRDLAEELKRLLKELREKGV >3plus1_GFP11_Key_Nterm_125 (sequence number 26726) SEKEDAARKLRKLVEELTREYEELVKKLERLIEEIEKVSEESVRKLEKLLAEISEEVR >3plus1_GFP11_Key_Nterm_126 (sequence number 26727) SKAEEIAEKLDRLLEENRRALEEITTRLDDLLRRNKDALRKVMEKLKRLLDDLRRGGI >3plus1_GFP11_Key_Nterm_127 (sequence number 26728) SEKEDAARKLRKLVEELTREYEELVKKLERLIEEIEKVSEESVRKLEKLLAEISEEVR >3plus1_GFP11_Key_Nterm_128 (sequence number 26729) SKAEEIAEKLDRLLEENRRALEEITTRLDDLLRRNKDALRKVMEKLKRLLDDLRRGGI >3plus1_GFP11_Key_Nterm_129 (sequence number 26730) SKEETLRKEAEDLLRRLEELTRRLEKKARELLERAKKLSRDLAEELKRLLKELREKGV >3plus1_GFP11_Key_Nterm_130 (sequence number 26731) SRVEELKKLIEDILRISREVVERIKRVAEDIHRINRRVLDDLRKLIEDILRTVEEILA >3plus1_GFP11_Key_Nterm_131 (sequence number 26732) SKEETLRKEAEDLLRRLEELTRRLEKKARELLERAKKLSRDLAEELKRLLKELREKGV >3plus1_GFP11_Key_Nterm_132 (sequence number 26733) SKAEEIAEKLDRLLEENRRALEEITTRLDDLLRRNKDALRKVMEKLKRLLDDLRRGGI >3plus1_GFP11_Key_Nterm_133 (sequence number 26734) SKEETLRKEAEDLLRRLEELTRRLEKKARELLERAKKLSRDLAEELKRLLKELREKGV >3plus1_GFP11_Key_Nterm_134 (sequence number 26735) SEKEDAARKLRKLVEELTREYEELVKKLERLIEEIEKVSEESVRKLEKLLAEISEEVR >3plus1_GFP11_Key_Nterm_135 (sequence number 26736) SKEETLRKEAEDLLRRLEELTRRLEKKARELLERAKKLSRDLAEELKRLLKELREKGV >3plus1_GFP11_Key_Nterm_136 (sequence number 26737) SERETVKRRLEELLKEVKRTLDKLKEEHDRLLEDVRRVVEELKREHDKLLKEVKDSGV >3plus1_GFP11_Key_Nterm_137 (sequence number 26738) SEKEDAARKLRKLVEELTREYEELVKKLERLIEEIEKVSEESVRKLEKLLAEISEEVR >3plus1_GFP11_Key_Nterm_138 (sequence number 26739) SKEETLRKEAEDLLRRLEELTRRLEKKARELLERAKKLSRDLAEELKRLLKELREKGV >3plus1_GFP11_Key_Nterm_139 (sequence number 26740) KEREEVKEKLDRLLEEVEKTVRELKREHDELLKEVEKLVRDLKKEHDELLKKVKDDGV >3plus1_GFP11_Key_Nterm_140 (sequence number 26741) SREEVLRELEEVIEDNRRLLEELIEKSKKVLDESLKLIDELLRRLEEVLERVLRLLEE >2plus1_GFP11_Key_Cterm_1 (sequence number 26742) DEVVKRVRDLLDTVRRRNEKVNEDVKRMNDKLRRDNEDVIRRVEKLLRELEEKRRT >2plus1_GFP11_Key_Cterm_2 (sequence number 26743) SEDSVERIARELERNLDDLARVLKESEDDLAEILRRLKEVLEESERDLERVEREVRK >2plus1_GFP11_Key_Cterm_3 (sequence number 26744) SKELLEKAKAVVDEIKRLAEESLKRLEDLSRDHKRRAKELNDEIAKVVDELAKRAT >2plus1_GFP11_Key_Cterm_4 (sequence number 26745) SKEKIDRIIRELERILEEAKKKHEDVLRRLEDSLRRVAELLKAALDRLREIVDRLRR >2plus1_GFP11_Key_Cterm_5 (sequence number 26746) DEVVKRVRDLLDTVRRRNEKVNEDVKRMNDKLRRDNEDVIRRVEKLLRELEEKRRT >2plus1_GFP11_Key_Cterm_6 (sequence number 26747) DIKTLLDRVRKLAEEDAERLDRLRRESEELNERVRRVDKKLLEEIRRKAKKVEDDTR >2plus1_GFP11_Key_Cterm_7 (sequence number 26748) DAETLLRELEKLSRDNKELLKKIEKEIRDLIKEDKERNIELSERLRKLVEELKKKAT >2plus1_GFP11_Key_Cterm_8 (sequence number 26749) DEVVKRVRDLLDTVRRRNEKVNEDVKRMNDKLRRDNEDVIRRVEKLLRELEEKRRT >2plus1_GFP11_Key_Cterm_9 (sequence number 26750) DIKTLLDRVRKLAEEDAERLDRLRRESEELNERVRRVDKKLLEEIRRKAKKVEDDTR >2plus1_GFP11_Key_Cterm_10 (sequence number 26751) SEELSAEVKKLLDEVRKALARHKDENDKLLKEIEDSLRRHKEENDRLLEKLKESTR >2plus1_GFP11_Key_Cterm_11 (sequence number 26752) DADDVLARVEELAKRAHDENERLIREVEELVRAHNKRNKELVDEVKRLVEKVIEEER >2plus1_GFP11_Key_Cterm_12 (sequence number 26753) SKEKIDRIIRELERILEEAKKKHEDVLRRLEDSLRRVAELLKAALDRLREIVDRLRR >2plus1_GFP11_Key_Cterm_13 (sequence number 26754) DEVVKRVRDLLDTVRRRNEKVNEDVKRMNDKLRRDNEDVIRRVEKLLRELEEKRRT >2plus1_GFP11_Key_Cterm_14 (sequence number 26755) SEELSAEVKKLLDEVRKALARHKDENDKLLKEIEDSLRRHKEENDRLLEKLKESTR >2plus1_GFP11_Key_Cterm_15 (sequence number 26756) DAETVLRSAEDIVAKNRKLAEEVLRRVKKIVEENRKIASEVLDDVRKLVEDVLARAS >2plus1_GFP11_Key_Cterm_16 (sequence number 26757) DADDVLARVEELAKRAHDENERLIREVEELVRAHNKRNKELVDEVKRLVEKVIEEER >2plus1_GFP11_Key_Cterm_17 (sequence number 26758) SKEKIDRIIRELERILEEAKKKHEDVLRRLEDSLRRVAELLKAALDRLREIVDRLRR >2plus1_GFP11_Key_Cterm_18 (sequence number 26759) DEEKLKDLIRKLRDILRRAAEAHKKLIDDARESLERAKREHEKLIDRLKKILEELER >2plus1_GFP11_Key_Cterm_19 (sequence number 26760) DIKTLLDRVRKLAEEDAERLDRLRRESEELNERVRRVDKKLLEEIRRKAKKVEDDTR >2plus1_GFP11_Key_Cterm_20 (sequence number 26761) DATRVIEEAKRILDEARKLNEETIRRSEELVRRIERVIEEIIKRSEKLLEDVARESK >2plus1_GFP11_Key_Cterm_21 (sequence number 26762) SEELSAEVKKLLDEVRKALARHKDENDKLLKEIEDSLRRHKEENDRLLEKLKESTR >2plus1_GFP11_Key_Cterm_22 (sequence number 26763) DAETVLRSAEDIVAKNRKLAEEVLRRVKKIVEENRKIASEVLDDVRKLVEDVLARAS >2plus1_GFP11_Key_Cterm_23 (sequence number 26764) DADDVLARVEELAKRAHDENERLIREVEELVRAHNKRNKELVDEVKRLVEKVIEEER >2plus1_GFP11_Key_Cterm_24 (sequence number 26765) SKELLEKAKAVVDEIKRLAEESLKRLEDLSRDHKRRAKELNDEIAKVVDELAKRAT >2plus1_GFP11_Key_Cterm_25 (sequence number 26766) DEEVLKKLAEIVRRVKEENRKVNEEVEKRLRELEEENKKVIEDLKSTVEELVERLR >2plus1_GFP11_Key_Cterm_26 (sequence number 26767) DEEVLKKLAEIVRRVKEENRKVNEEVEKRLRELEEENKKVIEDLKSTVEELVERLR >2plus1_GFP11_Key_Cterm_27 (sequence number 26768) DKLLKEARDLIREIEKRLEELLKRVEKLTEDAKRDLERSNREHKELADRIKETAR >2plus1_GFP11_Key_Cterm_28 (sequence number 26769) DKDSARELERIVKENAELAERVFREVEKIVRENTKLAEDSVRELKRLVEELKKRAK >2plus1_GFP11_Key_Cterm_29 (sequence number 26770) SKEKIDRIIRELERILEEAKKKHEDVLRRLEDSLRRVAELLKAALDRLREIVDRLRR >2plus1_GFP11_Key_Cterm_30 (sequence number 26771) DEEKLKDLIRKLRDILRRAAEAHKKLIDDARESLERAKREHEKLIDRLKKILEELER >2plus1_GFP11_Key_Cterm_31 (sequence number 26772) DEVVKRVRDLLDTVRRRNEKVNEDVKRMNDKLRRDNEDVIRRVEKLLRELEEKRRT >2plus1_GFP11_Key_Cterm_32 (sequence number 26773) DEEVLRTLEEIIRRLTKELEDVLREYERELRRLEEENKRVIDKTEEEIRRLADRLRR >2plus1_GFP11_Key_Cterm_33 (sequence number 26774) DERILRELEERVKELEKEAREILKRSEDETDKLREKAERILEDLERANRRTMDEARR >2plus1_GFP11_Key_Cterm_34 (sequence number 26775) SEELSAEVKKLLDEVRKALARHKDENDKLLKEIEDSLRRHKEENDRLLEKLKESTR >2plus1_GFP11_Key_Cterm_35 (sequence number 26776) LPEEVLRELEELLKESEERIKRIEEEIKKIIDKSREDIKRVLEEIERLNAKAADDLRK >2plus1_GFP11_Key_Cterm_36 (sequence number 26777) DADDVLARVEELAKRAHDENERLIREVEELVRAHNKRNKELVDEVKRLVEKVIEEER >2plus1_GFP11_Key_Cterm_37 (sequence number 26778) DEEVLKKLAEIVRRVKEENRKVNEEVEKRLRELEEENKKVIEDLKSTVEELVERLR >2plus1_GFP11_Key_Cterm_38 (sequence number 26779) DKLLKEARDLIREIEKRLEELLKRVEKLTEDAKRDLERSNREHKELADRIKETAR >2plus1_GFP11_Key_Cterm_39 (sequence number 26780) DEEVLRTLEEIIRRLTKELEDVLREYERELRRLEEENKRVIDKTEEEIRRLADRLRR >2plus1_GFP11_Key_Cterm_40 (sequence number 26781) DRRIEKVLKEIEEKIREVIKEWERVHREVEELLKRLIDENRKVLDEIRKLLEEKSK >2plus1_GFP11_Key_Cterm_41 (sequence number 26782) DERILRELEERVKELEKEAREILKRSEDETDKLREKAERILEDLERANRRTMDEARR >2plus1_GFP11_Key_Cterm_42 (sequence number 26783) SEELSAEVKKLLDEVRKALARHKDENDKLLKEIEDSLRRHKEENDRLLEKLKESTR >2plus1_GFP11_Key_Cterm_43 (sequence number 26784) DEEVLKKLAEIVRRVKEENRKVNEEVEKRLRELEEENKKVIEDLKSTVEELVERLR >2plus1_GFP11_Key_Cterm_44 (sequence number 26785) SKEKIDRIIRELERILEEAKKKHEDVLRRLEDSLRRVAELLKAALDRLREIVDRLRR >2plus1_GFP11_Key_Cterm_45 (sequence number 26786) DRRIEKVLKEIEEKIREVIKEWERVHREVEELLKRLIDENRKVLDEIRKLLEEKSK >2plus1_GFP11_Key_Cterm_46 (sequence number 26787) TLRELARSIRKLSAENKERLKELLRELKKLSDENKERIKKLLSDAEKIIEDVARRAK >2plus1_GFP11_Key_Cterm_47 (sequence number 26788) DERILRELEERVKELEKEAREILKRSEDETDKLREKAERILEDLERANRRTMDEARR >2plus1_GFP11_Key_Cterm_48 (sequence number 26789) EKLKELRDVIAEVAKRIDELDEYTRESIRRAKKEIERLNRETKKVIEEVVKRIEEERK >2plus1_GFP11_Key_Cterm_49 (sequence number 26790) DERVREELKKLLTRVEEEHRKVLETDKKILKEAHKESKEVNDRDRELLERLEESVR >2plus1_GFP11_Key_Cterm_50 (sequence number 26791) DADDVLARVEELAKRAHDENERLIREVEELVRAHNKRNKELVDEVKRLVEKVIEEER >2plus1_GFP11_Key_Cterm_51 (sequence number 26792) TVKRLLDELRELLERLKRTIEELLKRNRDLLADAEEKARRLLEENRKLLKAARDTAT >2plus1_GFP11_Key_Cterm_52 (sequence number 26793) DEEVLKKLAEIVRRVKEENRKVNEEVEKRLRELEEENKKVIEDLKSTVEELVERLR >2plus1_GFP11_Key_Cterm_53 (sequence number 26794) SKEKIDRIIRELERILEEAKKKHEDVLRRLEDSLRRVAELLKAALDRLREIVDRLRR >2plus1_GFP11_Key_Cterm_54 (sequence number 26795) DERILRELEERVKELEKEAREILKRSEDETDKLREKAERILEDLERANRRTMDEARR >2plus1_GFP11_Key_Cterm_55 (sequence number 26796) DATRVIEEAKRILDEARKLNEETIRRSEELVRRIERVIEEIIKRSEKLLEDVARESK >2plus1_GFP11_Key_Cterm_56 (sequence number 26797) EAAREIIKRLREVNKRTKEKLDELIKHSEEVLERVKRLIDELRKHSEEVLEDLRRRAK >2plus1_GFP11_Key_Cterm_57 (sequence number 26798) EKLKELRDVIAEVAKRIDELDEYTRESIRRAKKEIERLNRETKKVIEEVVKRIEEERK >2plus1_GFP11_Key_Cterm_58 (sequence number 26799) ELLRRIKKLLDEIKKAIEDSSREIKRLLEESERVMKRSSEDIKRTLDDTRRVVEEVRR >2plus1_GFP11_Key_Cterm_59 (sequence number 26800) SKAIKDVRDIVKKVKDELKEWRDRNKELVDRLSEELKEWLKDVERVLKELTDKDR >2plus1_GFP11_Key_Cterm_60 (sequence number 26801) DERVREELKKLLTRVEEEHRKVLETDKKILKEAHKESKEVNDRDRELLERLEESVR >2plus1_GFP11_Key_Cterm_61 (sequence number 26802) DIDKLLKELRDLVEKIKKDLKELLERYEEIVRRIKELLKDLNREAEEVVRRLKEELR >2plus1_GFP11_Key_Cterm_62 (sequence number 26803) DADDVLARVEELAKRAHDENERLIREVEELVRAHNKRNKELVDEVKRLVEKVIEEER >2plus1_GFP11_Key_Cterm_63 (sequence number 26804) DEEVLKKLAEIVRRVKEENRKVNEEVEKRLRELEEENKKVIEDLKSTVEELVERLR >2plus1_GFP11_Key_Cterm_64 (sequence number 26805) EREEELKEVADRVKEKLDRLNRENEKSSEELKRELDKINDENRETSERLKREIDETTR >2plus1_GFP11_Key_Cterm_65 (sequence number 26806) SKEKIDRIIRELERILEEAKKKHEDVLRRLEDSLRRVAELLKAALDRLREIVDRLRR >2plus1_GFP11_Key_Cterm_66 (sequence number 26807) SKEKIDRIIRELERILEEAKKKHEDVLRRLEDSLRRVAELLKAALDRLREIVDRLRR >2plus1_GFP11_Key_Cterm_67 (sequence number 26808) TKDLLDENSKRSNEISREVKKDLERTVRENKKIVDEVAKALEDTVDKNRRIVEEVTT >2plus1_GFP11_Key_Cterm_68 (sequence number 26809) DEVVKRVRDLLDTVRRRNEKVNEDVKRMNDKLRRDNEDVIRRVEKLLRELEEKRRT >2plus1_GFP11_Key_Cterm_69 (sequence number 26810) DRRIEKVLKEIEEKIREVIKEWERVHREVEELLKRLIDENRKVLDEIRKLLEEKSK >2plus1_GFP11_Key_Cterm_70 (sequence number 26811) ELLRRIKKLLDEIKKAIEDSSREIKRLLEESERVMKRSSEDIKRTLDDTRRVVEEVRR >2plus1_GFP11_Key_Cterm_71 (sequence number 26812) SEELSAEVKKLLDEVRKALARHKDENDKLLKEIEDSLRRHKEENDRLLEKLKESTR >2plus1_GFP11_Key_Cterm_72 (sequence number 26813) DIDKLLKELRDLVEKIKKDLKELLERYEEIVRRIKELLKDLNREAEEVVRRLKEELR >2plus1_GFP11_Key_Cterm_73 (sequence number 26814) DADDVLARVEELAKRAHDENERLIREVEELVRAHNKRNKELVDEVKRLVEKVIEEER >2plus1_GFP11_Key_Cterm_74 (sequence number 26815) DEEVLKKLAEIVRRVKEENRKVNEEVEKRLRELEEENKKVIEDLKSTVEELVERLR >2plus1_GFP11_Key_Cterm_75 (sequence number 26816) DEEVLKKLAEIVRRVKEENRKVNEEVEKRLRELEEENKKVIEDLKSTVEELVERLR >2plus1_GFP11_Key_Cterm_76 (sequence number 26817) SKEKIDRIIRELERILEEAKKKHEDVLRRLEDSLRRVAELLKAALDRLREIVDRLRR >2plus1_GFP11_Key_Cterm_77 (sequence number 26818) SEELREELKKLERKIEKVAKEIHDHDKEVTERLEDLLRRITEHARKSDREIEETAR >2plus1_GFP11_Key_Cterm_78 (sequence number 26819) DRRIEKVLKEIEEKIREVIKEWERVHREVEELLKRLIDENRKVLDEIRKLLEEKSK >2plus1_GFP11_Key_Cterm_79 (sequence number 26820) DATRVIEEAKRILDEARKLNEETIRRSEELVRRIERVIEEIIKRSEKLLEDVARESK >2plus1_GFP11_Key_Cterm_80 (sequence number 26821) EKLKELRDVIAEVAKRIDELDEYTRESIRRAKKEIERLNRETKKVIEEVVKRIEEERK >2plus1_GFP11_Key_Cterm_81 (sequence number 26822) ELLRRIKKLLDEIKKAIEDSSREIKRLLEESERVMKRSSEDIKRTLDDTRRVVEEVRR >2plus1_GFP11_Key_Cterm_82 (sequence number 26823) DIDKLLKELRDLVEKIKKDLKELLERYEEIVRRIKELLKDLNREAEEVVRRLKEELR >2plus1_GFP11_Key_Cterm_83 (sequence number 26824) EAKKKLDEVLERAKRTIDRLLETSDRSLEKVEADLRRLNEELDRSLERAERTIRELAK >2plus1_GFP11_Key_Cterm_84 (sequence number 26825) DEEVLKKLAEIVRRVKEENRKVNEEVEKRLRELEEENKKVIEDLKSTVEELVERLR >2plus1_GFP11_Key_Cterm_85 (sequence number 26826) DEEVLKKLAEIVRRVKEENRKVNEEVEKRLRELEEENKKVIEDLKSTVEELVERLR >2plus1_GFP11_Key_Cterm_86 (sequence number 26827) DRRIEKVLKEIEEKIREVIKEWERVHREVEELLKRLIDENRKVLDEIRKLLEEKSK >2plus1_GFP11_Key_Cterm_87 (sequence number 26828) DERILRELEERVKELEKEAREILKRSEDETDKLREKAERILEDLERANRRTMDEARR >2plus1_GFP11_Key_Cterm_88 (sequence number 26829) DLKRVEERAREVSRRNEESMRRVKEDADRVSEANKEVLDRVREEVKRLIEVRETLR >2plus1_GFP11_Key_Cterm_89 (sequence number 26830) EKLKELRDVIAEVAKRIDELDEYTRESIRRAKKEIERLNRETKKVIEEVVKRIEEERK >2plus1_GFP11_Key_Cterm_90 (sequence number 26831) ELLRRIKKLLDEIKKAIEDSSREIKRLLEESERVMKRSSEDIKRTLDDTRRVVEEVRR >2plus1_GFP11_Key_Cterm_91 (sequence number 26832) ELLRRIKKLLDEIKKAIEDSSREIKRLLEESERVMKRSSEDIKRTLDDTRRVVEEVRR >2plus1_GFP11_Key_Cterm_92 (sequence number 26833) LPEEVLRELEELLKESEERIKRIEEEIKKIIDKSREDIKRVLEEIERLNAKAADDLRK >2plus1_GFP11_Key_Cterm_93 (sequence number 26834) EAKKKLDEVLERAKRTIDRLLETSDRSLEKVEADLRRLNEELDRSLERAERTIRELAK >2plus1_GFP11_Key_Cterm_94 (sequence number 26835) DEEVLKKLAEIVRRVKEENRKVNEEVEKRLRELEEENKKVIEDLKSTVEELVERLR >2plus1_GFP11_Key_Cterm_95 (sequence number 26836) DKLLKEARDLIREIEKRLEELLKRVEKLTEDAKRDLERSNREHKELADRIKETAR >2plus1_GFP11_Key_Cterm_96 (sequence number 26837) DKLLKEARDLIREIEKRLEELLKRVEKLTEDAKRDLERSNREHKELADRIKETAR >2plus1_GFP11_Key_Cterm_97 (sequence number 26838) DIVRKIERIVETIEREVRESVKKVEEIARDIRRKVDESVKNVEKLLRDVDKKARDRKK >2plus1_GFP11_Key_Cterm_98 (sequence number 26839) DEIKRIVDEVRERLKRIVDENAKIVEDARRALEKIVKENEEILRRLKKELRELRK >2plus1_GFP11_Key_Cterm_99 (sequence number 26840) DRRIEKVLKEIEEKIREVIKEWERVHREVEELLKRLIDENRKVLDEIRKLLEEKSK >2plus1_GFP11_Key_Cterm_100 (sequence number 26841) DLKRVEERAREVSRRNEESMRRVKEDADRVSEANKEVLDRVREEVKRLIEVRETLR >2plus1_GFP11_Key_Cterm_101 (sequence number 26842) DATRVIEEAKRILDEARKLNEETIRRSEELVRRIERVIEEIIKRSEKLLEDVARESK >2plus1_GFP11_Key_Cterm_102 (sequence number 26843) DAETIERVVRELLEENKEVLRKTEEAVKRSTETNKRLLEASKEVADRLRERIKEAAK >2plus1_GFP11_Key_Cterm_103 (sequence number 26844) EKLKELRDVIAEVAKRIDELDEYTRESIRRAKKEIERLNRETKKVIEEVVKRIEEERK >2plus1_GFP11_Key_Cterm_104 (sequence number 26845) ELLRRIKKLLDEIKKAIEDSSREIKRLLEESERVMKRSSEDIKRTLDDTRRVVEEVRR >2plus1_GFP11_Key_Cterm_105 (sequence number 26846) DEVVERAERISEENKRRVEDVARKSKELVEDVRRHSEEVVRRVEELVKEVEERVR >2plus1_GFP11_Key_Cterm_106 (sequence number 26847) EAKKKLDEVLERAKRTIDRLLETSDRSLEKVEADLRRLNEELDRSLERAERTIRELAK >2plus1_GFP11_Key_Cterm_107 (sequence number 26848) TAERARETLKRLLDENRDRSKKVKEEIRRILEDLTRTTERVKREIAKLLKELEDTAR >2plus1_GFP11_Key_Cterm_108 (sequence number 26849) EAVRRLKEILERLKEEVRRSLEELRKEVERLKKEVEDSLRELKKSLEEWVKSLEEATR >2plus1_GFP11_Key_Cterm_109 (sequence number 26850) DERILRELEERVKELEKEAREILKRSEDETDKLREKAERILEDLERANRRTMDEARR >2plus1_GFP11_Key_Cterm_110 (sequence number 26851) DATRVIEEAKRILDEARKLNEETIRRSEELVRRIERVIEEIIKRSEKLLEDVARESK >2plus1_GFP11_Key_Cterm_111 (sequence number 26852) DKARKVAEVAEKVLRDIDKLDRESKEAFRATNEEIAKLDEDTARVAERVKKAIEDLAK >2plus1_GFP11_Key_Cterm_112 (sequence number 26853) EKLKELRDVIAEVAKRIDELDEYTRESIRRAKKEIERLNRETKKVIEEVVKRIEEERK >2plus1_GFP11_Key_Cterm_113 (sequence number 26854) ELLRRIKKLLDEIKKAIEDSSREIKRLLEESERVMKRSSEDIKRTLDDTRRVVEEVRR >2plus1_GFP11_Key_Cterm_114 (sequence number 26855) DKVERVVREVEKLHEEDRKRLEESTRSVRKLLEELKRELEKSTRSVKALVDELRERVR >2plus1_GFP11_Key_Cterm_115 (sequence number 26856) DEVVERAERISEENKRRVEDVARKSKELVEDVRRHSEEVVRRVEELVKEVEERVR >2plus1_GFP11_Key_Cterm_116 (sequence number 26857) EAKKKLDEVLERAKRTIDRLLETSDRSLEKVEADLRRLNEELDRSLERAERTIRELAK >2plus1_GFP11_Key_Cterm_117 (sequence number 26858) TAERARETLKRLLDENRDRSKKVKEEIRRILEDLTRTTERVKREIAKLLKELEDTAR >2plus1_GFP11_Key_Cterm_118 (sequence number 26859) DERILRELEERVKELEKEAREILKRSEDETDKLREKAERILEDLERANRRTMDEARR >2plus1_GFP11_Key_Cterm_119 (sequence number 26860) DERILRELEERVKELEKEAREILKRSEDETDKLREKAERILEDLERANRRTMDEARR >2plus1_GFP11_Key_Cterm_120 (sequence number 26861) DLKRVEERAREVSRRNEESMRRVKEDADRVSEANKEVLDRVREEVKRLIEVRETLR >2plus1_GFP11_Key_Cterm_121 (sequence number 26862) DKARKVAEVAEKVLRDIDKLDRESKEAFRATNEEIAKLDEDTARVAERVKKAIEDLAK >2plus1_GFP11_Key_Cterm_122 (sequence number 26863) ELLRRIKKLLDEIKKAIEDSSREIKRLLEESERVMKRSSEDIKRTLDDTRRVVEEVRR >2plus1_GFP11_Key_Cterm_123 (sequence number 26864) SKAIKDVRDIVKKVKDELKEWRDRNKELVDRLSEELKEWLKDVERVLKELTDKDR >2plus1_GFP11_Key_Cterm_124 (sequence number 26865) DKVERVVREVEKLHEEDRKRLEESTRSVRKLLEELKRELEKSTRSVKALVDELRERVR >2plus1_GFP11_Key_Cterm_125 (sequence number 26866) DIDKLLKELRDLVEKIKKDLKELLERYEEIVRRIKELLKDLNREAEEVVRRLKEELR >2plus1_GFP11_Key_Cterm_126 (sequence number 26867) EAKKKLDEVLERAKRTIDRLLETSDRSLEKVEADLRRLNEELDRSLERAERTIRELAK >2plus1_GFP11_Key_Cterm_127 (sequence number 26868) DKLLKEARDLIREIEKRLEELLKRVEKLTEDAKRDLERSNREHKELADRIKETAR >2plus1_GFP11_Key_Cterm_128 (sequence number 26869) TAERARETLKRLLDENRDRSKKVKEEIRRILEDLTRTTERVKREIAKLLKELEDTAR >2plus1_GFP11_Key_Cterm_129 (sequence number 26870) DEIKRIVDEVRERLKRIVDENAKIVEDARRALEKIVKENEEILRRLKKELRELRK >2plus1_GFP11_Key_Cterm_130 (sequence number 26871) DRIEEELKRLIDTLREKNREVEKRARDSNRDLKRTNDEIAKEVRELIKKLREDLK >2plus1_GFP11_Key_Cterm_131 (sequence number 26872) DERILRELEERVKELEKEAREILKRSEDETDKLREKAERILEDLERANRRTMDEARR >2plus1_GFP11_Key_Cterm_132 (sequence number 26873) DAETIERVVRELLEENKEVLRKTEEAVKRSTETNKRLLEASKEVADRLRERIKEAAK >2plus1_GFP11_Key_Cterm_133 (sequence number 26874) DKARKVAEVAEKVLRDIDKLDRESKEAFRATNEEIAKLDEDTARVAERVKKAIEDLAK >2plus1_GFP11_Key_Cterm_134 (sequence number 26875) DKVERVVREVEKLHEEDRKRLEESTRSVRKLLEELKRELEKSTRSVKALVDELRERVR >2plus1_GFP11_Key_Cterm_135 (sequence number 26876) DIERILRELEAVLKKLTDESERLNREVERVSRDTKKKSKELNEELKAVLDEVKRKAD >2plus1_GFP11_Key_Cterm_136 (sequence number 26877) DEVVERAERISEENKRRVEDVARKSKELVEDVRRHSEEVVRRVEELVKEVEERVR >2plus1_GFP11_Key_Cterm_137 (sequence number 26878) EAKKKLDEVLERAKRTIDRLLETSDRSLEKVEADLRRLNEELDRSLERAERTIRELAK >2plus1_GFP11_Key_Cterm_138 (sequence number 26879) TAERARETLKRLLDENRDRSKKVKEEIRRILEDLTRTTERVKREIAKLLKELEDTAR >2plus1_GFP11_Key_Cterm_139 (sequence number 26880) DEIKRIVDEVRERLKRIVDENAKIVEDARRALEKIVKENEEILRRLKKELRELRK >2plus1_GFP11_Key_Cterm_140 (sequence number 26881) DRRIEKVLKEIEEKIREVIKEWERVHREVEELLKRLIDENRKVLDEIRKLLEEKSK >2plus1_GFP11_Key_Cterm_141 (sequence number 26882) DLKRVEERAREVSRRNEESMRRVKEDADRVSEANKEVLDRVREEVKRLIEVRETLR >2plus1_GFP11_Key_Cterm_142 (sequence number 26883) DAETIERVVRELLEENKEVLRKTEEAVKRSTETNKRLLEASKEVADRLRERIKEAAK >2plus1_GFP11_Key_Cterm_143 (sequence number 26884) DKARKVAEVAEKVLRDIDKLDRESKEAFRATNEEIAKLDEDTARVAERVKKAIEDLAK >2plus1_GFP11_Key_Cterm_144 (sequence number 26885) ELLRRIKKLLDEIKKAIEDSSREIKRLLEESERVMKRSSEDIKRTLDDTRRVVEEVRR >2plus1_GFP11_Key_Cterm_145 (sequence number 26886) DKVERVVREVEKLHEEDRKRLEESTRSVRKLLEELKRELEKSTRSVKALVDELRERVR >2plus1_GFP11_Key_Cterm_146 (sequence number 26887) EAKKKLDEVLERAKRTIDRLLETSDRSLEKVEADLRRLNEELDRSLERAERTIRELAK >2plus1_GFP11_Key_Cterm_147 (sequence number 26888) TAERARETLKRLLDENRDRSKKVKEEIRRILEDLTRTTERVKREIAKLLKELEDTAR >2plus1_GFP11_Key_Cterm_148 (sequence number 26889) DEIKRIVDEVRERLKRIVDENAKIVEDARRALEKIVKENEEILRRLKKELRELRK >2plus1_GFP11_Key_Cterm_149 (sequence number 26890) DKARKVAEVAEKVLRDIDKLDRESKEAFRATNEEIAKLDEDTARVAERVKKAIEDLAK >2plus1_GFP11_Key_Cterm_150 (sequence number 26891) DEVTKVKKVADDVLAEIKKLDDETRRVIEDTNKKIADLDKATRDVVRKVLEEVKKLEK >2plus1_GFP11_Key_Cterm_151 (sequence number 26892) DIDKLLKELRDLVEKIKKDLKELLERYEEIVRRIKELLKDLNREAEEVVRRLKEELR >2plus1_GFP11_Key_Cterm_152 (sequence number 26893) TAERARETLKRLLDENRDRSKKVKEEIRRILEDLTRTTERVKREIAKLLKELEDTAR >2plus1_GFP11_Key_Cterm_153 (sequence number 26894) DEIKRIVDEVRERLKRIVDENAKIVEDARRALEKIVKENEEILRRLKKELRELRK >2plus1_GFP11_Key_Cterm_154 (sequence number 26895) RLVREVEDLVRRLVRRSEKSNEEVKRTVEELVRRMEESNDRVRDLVRRLVEELKRAVD >2plus1_GFP11_Key_Cterm_155 (sequence number 26896) DKARKVAEVAEKVLRDIDKLDRESKEAFRATNEEIAKLDEDTARVAERVKKAIEDLAK >2plus1_GFP11_Key_Cterm_156 (sequence number 26897) EAKKKLDEVLERAKRTIDRLLETSDRSLEKVEADLRRLNEELDRSLERAERTIRELAK >2plus1_GFP11_Key_Cterm_157 (sequence number 26898) RLVREVEDLVRRLVRRSEKSNEEVKRTVEELVRRMEESNDRVRDLVRRLVEELKRAVD >2plus1_GFP11_Key_Cterm_158 (sequence number 26899) DKARKVAEVAEKVLRDIDKLDRESKEAFRATNEEIAKLDEDTARVAERVKKAIEDLAK >2plus1_GFP11_Key_Cterm_159 (sequence number 26900) DEVTKVKKVADDVLAEIKKLDDETRRVIEDTNKKIADLDKATRDVVRKVLEEVKKLEK >2plus1_GFP11_Key_Cterm_160 (sequence number 26901) TAERARETLKRLLDENRDRSKKVKEEIRRILEDLTRTTERVKREIAKLLKELEDTAR >2plus1_GFP11_Key_Cterm_161 (sequence number 26902) DLKRVEERAREVSRRNEESMRRVKEDADRVSEANKEVLDRVREEVKRLIEVRETLR >2plus1_GFP11_Key_Cterm_162 (sequence number 26903) DKARKVAEVAEKVLRDIDKLDRESKEAFRATNEEIAKLDEDTARVAERVKKAIEDLAK >2plus1_GFP11_Key_Cterm_163 (sequence number 26904) DEVTKVKKVADDVLAEIKKLDDETRRVIEDTNKKIADLDKATRDVVRKVLEEVKKLEK >2plus1_GFP11_Key_Cterm_164 (sequence number 26905) DKVERVVREVEKLHEEDRKRLEESTRSVRKLLEELKRELEKSTRSVKALVDELRERVR >2plus1_GFP11_Key_Cterm_165 (sequence number 26906) EAKKKLDEVLERAKRTIDRLLETSDRSLEKVEADLRRLNEELDRSLERAERTIRELAK >2plus1_GFP11_Key_Cterm_166 (sequence number 26907) TAERARETLKRLLDENRDRSKKVKEEIRRILEDLTRTTERVKREIAKLLKELEDTAR >2plus1_GFP11_Key_Cterm_167 (sequence number 26908) TAERARETLKRLLDENRDRSKKVKEEIRRILEDLTRTTERVKREIAKLLKELEDTAR >2plus1_GFP11_Key_Cterm_168 (sequence number 26909) DKARKVAEVAEKVLRDIDKLDRESKEAFRATNEEIAKLDEDTARVAERVKKAIEDLAK >2plus1_GFP11_Key_Cterm_169 (sequence number 26910) DEVTKVKKVADDVLAEIKKLDDETRRVIEDTNKKIADLDKATRDVVRKVLEEVKKLEK >2plus1_GFP11_Key_Cterm_170 (sequence number 26911) DKVERVVREVEKLHEEDRKRLEESTRSVRKLLEELKRELEKSTRSVKALVDELRERVR >2plus1_GFP11_Key_Cterm_171 (sequence number 26912) TAERARETLKRLLDENRDRSKKVKEEIRRILEDLTRTTERVKREIAKLLKELEDTAR >2plus1_GFP11_Key_Cterm_172 (sequence number 26913) DKARKVAEVAEKVLRDIDKLDRESKEAFRATNEEIAKLDEDTARVAERVKKAIEDLAK >2plus1_GFP11_Key_Cterm_173 (sequence number 26914) RLVREVEDLVRRLVRRSEKSNEEVKRTVEELVRRMEESNDRVRDLVRRLVEELKRAVD >2plus1_GFP11_Key_Nterm_174 (sequence number 26915) SESDEVIRDLARLLDELARHVDDSVRRMDEVVKRSTREADELAKRLDELVKEVEKKPG >2plus1_GFP11_Key_Nterm_175 (sequence number 26916) SRAETVLKEVTDKIKKLADSSDELLRRNKENIDELKKSSEELLRRLTKAIEEIEKGSV >2plus1_GFP11_Key_Nterm_176 (sequence number 26917) SVDEVLKEIEDALRRLKKEEVERVLKENEDELRRLEEEVRRVLKEDEELLESLKRGVGE >2plus1_GFP11_Key_Nterm_177 (sequence number 26918) SEVDEIIKELERLLAEIARENERIIRESRKLADEVRKRNEDAIRKLEELVARLADAVR >2plus1_GFP11_Key_Nterm_178 (sequence number 26919) SEVDDVLRRLEELIKTLEDINAKSLEDIKKLIDDLAKILEDALRKHEKLIRELREAKK >2plus1_GFP11_Key_Nterm_179 (sequence number 26920) SEVDDVLRRLEELIKTLEDINAKSLEDIKKLIDDLAKILEDALRKHEKLIRELREAKK >2plus1_GFP11_Key_Nterm_180 (sequence number 26921) SRAETVLKEVTDKIKKLADSSDELLRRNKENIDELKKSSEELLRRLTKAIEEIEKGSV >2plus1_GFP11_Key_Nterm_181 (sequence number 26922) KEVEDAVKELEDLLRANEDKTRSIVEDMRASNKDLEDHSRASEEEVRKLLDDLRRAGV >2plus1_GFP11_Key_Nterm_182 (sequence number 26923) SEVDDVLRRLEELIKTLEDINAKSLEDIKKLIDDLAKILEDALRKHEKLIRELREAKK >2plus1_GFP11_Key_Nterm_183 (sequence number 26924) SESDEVIRDLARLLDELARHVDDSVRRMDEVVKRSTREADELAKRLDELVKEVEKKPG >2plus1_GFP11_Key_Nterm_184 (sequence number 26925) SESDDVIRKLRELLEELRTHVEKSIRDLRKILEDSTRHAKRSIEELERLLEEVRKKPG >2plus1_GFP11_Key_Nterm_185 (sequence number 26926) SRAETVLKEVTDKIKKLADSSDELLRRNKENIDELKKSSEELLRRLTKAIEEIEKGSV >2plus1_GFP11_Key_Nterm_186 (sequence number 26927) SEAEKAKETIDRLADRVRKLLEEIKRSLDDSRRKSKETVEENEKTLDRMRKEVDAAKR >2plus1_GFP11_Key_Nterm_187 (sequence number 26928) SEAEKAKETIDRLADRVRKLLEEIKRSLDDSRRKSKETVEENEKTLDRMRKEVDAAKR >2plus1_GFP11_Key_Nterm_188 (sequence number 26929) SEVEELIKRLAKVLKELVDKVRKVIEDTKELLERLKRRSEDHIRKLREVLKEAKDQPI >2plus1_GFP11_Key_Nterm_189 (sequence number 26930) SELEEIEKKVRELTKRHRELVERVRKTVKELIETNRRLLETLTERIKRVLEEVRDLER >2plus1_GFP11_Key_Nterm_190 (sequence number 26931) SSEERLRAVIEDLKRLAEESRKRHKELIDELAKAVERIERRHKKLLDEIKAVVDDIRR >2plus1_GFP11_Key_Nterm_191 (sequence number 26932) DEIRKVVKEITDLLKASNDKNRKVVEEIRDLLRKSKKLADELVERLRALVEDLRRRID >2plus1_GFP11_Key_Nterm_192 (sequence number 26933) STAETVAEEVERVLKHSDDLIKEVEDVNRRVEEEIKRVIRELEEENERLVAEVRKGVK >2plus1_GFP11_Key_Nterm_193 (sequence number 26934) SEVDEIIKELERLLAEIARENERIIRESRKLADEVRKRNEDAIRKLEELVARLADAVR >2plus1_GFP11_Key_Nterm_194 (sequence number 26935) SEIDEVLTRLRKISKDLNETSDRVNERARKIIDDIKKESKRVNDEAREIVERLKREID >2plus1_GFP11_Key_Nterm_195 (sequence number 26936) SEDEDLDRVAEKLAREHKKSVEEIKRVLKSADEESKKLVRDTERVIEEIKREVEEARR >2plus1_GFP11_Key_Nterm_196 (sequence number 26937) SSVEELLERLRRISEENKRRIEKLLREVEKVLRELKDRHRKLLKRVEEIIRKVKEEIK >2plus1_GFP11_Key_Nterm_197 (sequence number 26938) SAADEVVERMKELVATVKRENDEVVKELKKLVKELEDDNRRVVEESKKSVEDLARRVG >2plus1_GFP11_Key_Nterm_198 (sequence number 26939) SESDEVIRDLARLLDELARHVDDSVRRMDEVVKRSTREADELAKRLDELVKEVEKKPG >2plus1_GFP11_Key_Nterm_199 (sequence number 26940) KEVEDAVKELEDLLRANEDKTRSIVEDMRASNKDLEDHSRASEEEVRKLLDDLRRAGV >2plus1_GFP11_Key_Nterm_200 (sequence number 26941) DEIRKVVKEITDLLKASNDKNRKVVEEIRDLLRKSKKLADELVERLRALVEDLRRRID >2plus1_GFP11_Key_Nterm_201 (sequence number 26942) SRVEEIIEDLRRLLEEIRKENEDSIRRSKELLDRVKEINDTIIAELERLLKDIEKEVR >2plus1_GFP11_Key_Nterm_202 (sequence number 26943) SRVEEIIEDLRRLLEEIRKENEDSIRRSKELLDRVKEINDTIIAELERLLKDIEKEVR >2plus1_GFP11_Key_Nterm_203 (sequence number 26944) SEVDDVLRRLEELIKTLEDINAKSLEDIKKLIDDLAKILEDALRKHEKLIRELREAKK >2plus1_GFP11_Key_Nterm_204 (sequence number 26945) SKLEEVEKAVRKVIEDSRRVNEEVNRRSEEVVRELEKVHREVNDASRRVVEKARRVLK >2plus1_GFP11_Key_Nterm_205 (sequence number 26946) SESDEVIRDLARLLDELARHVDDSVRRMDEVVKRSTREADELAKRLDELVKEVEKKPG >2plus1_GFP11_Key_Nterm_206 (sequence number 26947) SESDEVIRDLARLLDELARHVDDSVRRMDEVVKRSTREADELAKRLDELVKEVEKKPG >2plus1_GFP11_Key_Nterm_207 (sequence number 26948) SESDDVIRKLRELLEELRTHVEKSIRDLRKILEDSTRHAKRSIEELERLLEEVRKKPG >2plus1_GFP11_Key_Nterm_208 (sequence number 26949) DEVRELLERNRRLLEEIKKTVKDLIRANEELLKRIEDDAKRLIDRNEELLDELEKGLS >2plus1_GFP11_Key_Nterm_209 (sequence number 26950) DEIRKVVKEITDLLKASNDKNRKVVEEIRDLLRKSKKLADELVERLRALVEDLRRRID >2plus1_GFP11_Key_Nterm_210 (sequence number 26951) SRAETVLKEVTDKIKKLADSSDELLRRNKENIDELKKSSEELLRRLTKAIEEIEKGSV >2plus1_GFP11_Key_Nterm_211 (sequence number 26952) DEEEDLERAIKKLLDENRELLKRIAEELRRLLEELRRLTEESADRLRRLLKELKDRGV >2plus1_GFP11_Key_Nterm_212 (sequence number 26953) DEIRKVVKEITDLLKASNDKNRKVVEEIRDLLRKSKKLADELVERLRALVEDLRRRID >2plus1_GFP11_Key_Nterm_213 (sequence number 26954) SKEDRLREELKKLLARLAEEIERLKRALEESNKDLKRTIDASEKHLRDVNEDVKRGGV >2plus1_GFP11_Key_Nterm_214 (sequence number 26955) SESDEVIRDLARLLDELARHVDDSVRRMDEVVKRSTREADELAKRLDELVKEVEKKPG >2plus1_GFP11_Key_Nterm_215 (sequence number 26956) SESDEVIRDLARLLDELARHVDDSVRRMDEVVKRSTREADELAKRLDELVKEVEKKPG >2plus1_GFP11_Key_Nterm_216 (sequence number 26957) SESDDVIRKLRELLEELRTHVEKSIRDLRKILEDSTRHAKRSIEELERLLEEVRKKPG >2plus1_GFP11_Key_Nterm_217 (sequence number 26958) SRVEEIIEDLRRLLEEIRKENEDSIRRSKELLDRVKEINDTIIAELERLLKDIEKEVR >2plus1_GFP11_Key_Nterm_218 (sequence number 26959) SEVDEIIKELERLLAEIARENERIIRESRKLADEVRKRNEDAIRKLEELVARLADAVR >2plus1_GFP11_Key_Nterm_219 (sequence number 26960) SSVEELLERLRRISEENKRRIEKLLREVEKVLRELKDRHRKLLKRVEEIIRKVKEEIK >2plus1_GFP11_Key_Nterm_220 (sequence number 26961) SELEEVLRRIEALVRKAHKENEDVLREIERLVRTAHRLNKKVDDDSAKIAEDLKRGGR >2plus1_GFP11_Key_Nterm_221 (sequence number 26962) SESDEVIRDLARLLDELARHVDDSVRRMDEVVKRSTREADELAKRLDELVKEVEKKPG >2plus1_GFP11_Key_Nterm_222 (sequence number 26963) SESDDVIRKLRELLEELRTHVEKSIRDLRKILEDSTRHAKRSIEELERLLEEVRKKPG >2plus1_GFP11_Key_Nterm_223 (sequence number 26964) SRAETVLKEVTDKIKKLADSSDELLRRNKENIDELKKSSEELLRRLTKAIEEIEKGSV >2plus1_GFP11_Key_Nterm_224 (sequence number 26965) DEVRELLERNRRLLEEIKKTVKDLIRANEELLKRIEDDAKRLIDRNEELLDELEKGLS >2plus1_GFP11_Key_Nterm_225 (sequence number 26966) STEEVLDEIRKLHKTLTEDIKRVLREIEELHRRTIEENKEVLDKIAEDYKRVIDDVRT >2plus1_GFP11_Key_Nterm_226 (sequence number 26967) SEIEKILKEIEDLARRDEEVSKKIVEDIRRLAKEVEDTSRDIVRKIEELAKRVLDRLR >2plus1_GFP11_Key_Nterm_227 (sequence number 26968) SEAERLEARARELLRANEELMDDLRAKAEELLKRNDRLVKEIEKKVREVLAAIEELKR >2plus1_GFP11_Key_Nterm_228 (sequence number 26969) DDLERAREEVADLIRKHEEKTRRILEESRRLNERHRELSARILDEIRKLAERIEELIK >2plus1_GFP11_Key_Nterm_229 (sequence number 26970) DEEEDLERAIKKLLDENRELLKRIAEELRRLLEELRRLTEESADRLRRLLKELKDRGV >2plus1_GFP11_Key_Nterm_230 (sequence number 26971) DEIRKVVKEITDLLKASNDKNRKVVEEIRDLLRKSKKLADELVERLRALVEDLRRRID >2plus1_GFP11_Key_Nterm_231 (sequence number 26972) DEIRKVVKEITDLLKASNDKNRKVVEEIRDLLRKSKKLADELVERLRALVEDLRRRID >2plus1_GFP11_Key_Nterm_232 (sequence number 26973) STAETVEKKVEEVIRENEKSMRESEEKVDRSTKRIEDVLRRLEETIRKTSDDIAKGVK >2plus1_GFP11_Key_Nterm_233 (sequence number 26974) SRVEEIIEDLRRLLEEIRKENEDSIRRSKELLDRVKEINDTIIAELERLLKDIEKEVR >2plus1_GFP11_Key_Nterm_234 (sequence number 26975) SRVEEIIEDLRRLLEEIRKENEDSIRRSKELLDRVKEINDTIIAELERLLKDIEKEVR >2plus1_GFP11_Key_Nterm_235 (sequence number 26976) REVEEMIKELEELLKDLKEKNERASKRNRELVRRLEEENKRVIEELKKLVKELEDLVR >2plus1_GFP11_Key_Nterm_236 (sequence number 26977) SEVDDVLRRLEELIKTLEDINAKSLEDIKKLIDDLAKILEDALRKHEKLIRELREAKK >2plus1_GFP11_Key_Nterm_237 (sequence number 26978) SKLEEVEKAVRKVIEDSRRVNEEVNRRSEEVVRELEKVHREVNDASRRVVEKARRVLK >2plus1_GFP11_Key_Nterm_238 (sequence number 26979) DEVEDVLRKIEKILDDHRKRIEKNSRDMARIIDEHRRKVEENSREMKKLVDDLKKAVD >2plus1_GFP11_Key_Nterm_239 (sequence number 26980) SSVEELLERLRRISEENKRRIEKLLREVEKVLRELKDRHRKLLKRVEEIIRKVKEEIK >2plus1_GFP11_Key_Nterm_240 (sequence number 26981) DEVEKVLEEIKRALDDLRKKVEESKREIKEALKAVEKHTRDSDTANKRTLAEIERGVK >2plus1_GFP11_Key_Nterm_241 (sequence number 26982) SESDEVIRDLARLLDELARHVDDSVRRMDEVVKRSTREADELAKRLDELVKEVEKKPG >2plus1_GFP11_Key_Nterm_242 (sequence number 26983) KEVEDAVKELEDLLRANEDKTRSIVEDMRASNKDLEDHSRASEEEVRKLLDDLRRAGV >2plus1_GFP11_Key_Nterm_243 (sequence number 26984) DEIRKVVKEITDLLKASNDKNRKVVEEIRDLLRKSKKLADELVERLRALVEDLRRRID >2plus1_GFP11_Key_Nterm_244 (sequence number 26985) STAETVAEEVERVLKHSDDLIKEVEDVNRRVEEEIKRVIRELEEENERLVAEVRKGVK >2plus1_GFP11_Key_Nterm_245 (sequence number 26986) SRVEEIIEDLRRLLEEIRKENEDSIRRSKELLDRVKEINDTIIAELERLLKDIEKEVR >2plus1_GFP11_Key_Nterm_246 (sequence number 26987) SEVDEIIKELERLLAEIARENERIIRESRKLADEVRKRNEDAIRKLEELVARLADAVR >2plus1_GFP11_Key_Nterm_247 (sequence number 26988) SEVDDVLRRLEELIKTLEDINAKSLEDIKKLIDDLAKILEDALRKHEKLIRELREAKK >2plus1_GFP11_Key_Nterm_248 (sequence number 26989) SKLEEVEKAVRKVIEDSRRVNEEVNRRSEEVVRELEKVHREVNDASRRVVEKARRVLK >2plus1_GFP11_Key_Nterm_249 (sequence number 26990) SAEEVKEELKRIATKLKEEIKENIRRLEESVEKIAKELAENIKRLEDILRDVKRGLRD >2plus1_GFP11_Key_Nterm_250 (sequence number 26991) SDVDRVLEEIRKLLEDLKRHSEKVSEENEDLLRANTELNKRVSEDNERLLEELKRLRE >2plus1_GFP11_Key_Nterm_251 (sequence number 26992) DEVEDVLRKIEKILDDHRKRIEKNSRDMARIIDEHRRKVEENSREMKKLVDDLKKAVD >2plus1_GFP11_Key_Nterm_252 (sequence number 26993) SSVEELLERLRRISEENKRRIEKLLREVEKVLRELKDRHRKLLKRVEEIIRKVKEEIK >2plus1_GFP11_Key_Nterm_253 (sequence number 26994) DREREVKKRLDEVRERIERLLRRVEEESRRVAEEIRRLIEEVRRRNKKVTEEIRELLK >2plus1_GFP11_Key_Nterm_254 (sequence number 26995) SRAETVLKEVTDKIKKLADSSDELLRRNKENIDELKKSSEELLRRLTKAIEEIEKGSV >2plus1_GFP11_Key_Nterm_255 (sequence number 26996) SEIEKILKEIEDLARRDEEVSKKIVEDIRRLAKEVEDTSRDIVRKIEELAKRVLDRLR >2plus1_GFP11_Key_Nterm_256 (sequence number 26997) SEAEKAKETIDRLADRVRKLLEEIKRSLDDSRRKSKETVEENEKTLDRMRKEVDAAKR >2plus1_GFP11_Key_Nterm_257 (sequence number 26998) SEVEELIKRLAKVLKELVDKVRKVIEDTKELLERLKRRSEDHIRKLREVLKEAKDQPI >2plus1_GFP11_Key_Nterm_258 (sequence number 26999) DEIRKVVKEITDLLKASNDKNRKVVEEIRDLLRKSKKLADELVERLRALVEDLRRRID >2plus1_GFP11_Key_Nterm_259 (sequence number 27000) DEIRKVVKEITDLLKASNDKNRKVVEEIRDLLRKSKKLADELVERLRALVEDLRRRID >2plus1_GFP11_Key_Nterm_260 (sequence number 27001) STAETVEKKVEEVIRENEKSMRESEEKVDRSTKRIEDVLRRLEETIRKTSDDIAKGVK >2plus1_GFP11_Key_Nterm_261 (sequence number 27002) SRVEEIIEDLRRLLEEIRKENEDSIRRSKELLDRVKEINDTIIAELERLLKDIEKEVR >2plus1_GFP11_Key_Nterm_262 (sequence number 27003) REVEEMIKELEELLKDLKEKNERASKRNRELVRRLEEENKRVIEELKKLVKELEDLVR >2plus1_GFP11_Key_Nterm_263 (sequence number 27004) DAVEEAEKLIRKVIADSEKLLRDLADLNAKSIRRSEKLVEDDRRANEDVIRKLEELRR >2plus1_GFP11_Key_Nterm_264 (sequence number 27004) SEVDDVLRRLEELIKTLEDINAKSLEDIKKLIDDLAKILEDALRKHEKLIRELREAKK >2plus1_GFP11_Key_Nterm_265 (sequence number 27006) SEIERVKKRLEELLAEVEESTRRLEERLKRLLEEAKRSSEEVEKELRRLLEAVRRGLS >2plus1_GFP11_Key_Nterm_266 (sequence number 27007) SDVDRVLEEIRKLLEDLKRHSEKVSEENEDLLRANTELNKRVSEDNERLLEELKRLRE >2plus1_GFP11_Key_Nterm_267 (sequence number 27008) DEVEDVLRKIEKILDDHRKRIEKNSRDMARIIDEHRRKVEENSREMKKLVDDLKKAVD >2plus1_GFP11_Key_Nterm_268 (sequence number 27009) SESDEVIRDLARLLDELARHVDDSVRRMDEVVKRSTREADELAKRLDELVKEVEKKPG >2plus1_GFP11_Key_Nterm_269 (sequence number 27010) SRAETVLKEVTDKIKKLADSSDELLRRNKENIDELKKSSEELLRRLTKAIEEIEKGSV >2plus1_GFP11_Key_Nterm_270 (sequence number 27011) DEEEDLERAIKKLLDENRELLKRIAEELRRLLEELRRLTEESADRLRRLLKELKDRGV >2plus1_GFP11_Key_Nterm_271 (sequence number 27012) DEIRKVVKEITDLLKASNDKNRKVVEEIRDLLRKSKKLADELVERLRALVEDLRRRID >2plus1_GFP11_Key_Nterm_272 (sequence number 27013) DAVEEAEKLIRKVIADSEKLLRDLADLNAKSIRRSEKLVEDDRRANEDVIRKLEELRR >2plus1_GFP11_Key_Nterm_273 (sequence number 27014) SEDEDLDRVAEKLAREHKKSVEEIKRVLKSADEESKKLVRDTERVIEEIKREVEEARR >2plus1_GFP11_Key_Nterm_274 (sequence number 27015) SKEDRLREELKKLLARLAEEIERLKRALEESNKDLKRTIDASEKHLRDVNEDVKRGGV >3plus1_Key_668_Nterm (sequence number 27,322) DEAKELLDEIRKAVKESEDRLEKLLRDYEKELRRLEKELRDLKRRIEEKLEELRRGSL >3plus1_Key_668_Cterm (sequence number 27,323) RGADALSRLLEELLRVVDDLIRVLKELIDKSRKVIEELLELLKRINEENLKVLAEIIK >3plus1_Key_668_Cterm (sequence number 27,324) SEKEKLLKESEEEVRRLRRTLEELLRKYREVLERLRKELREIEERVRDVVRRLKEVLD >3plus1_Key_668_Cterm (sequence number 27,325) SEKEDAARKLRKLVEELTREYEELVKKLERLIEEIEKVSEESVRKLEKLLAEISEEVR >3plus1_Key_668_Cterm (sequence number 27,326) EALRKLVELLVEVLRRLIRVNRELVKLLREVLERLLRILRESVKKLKRLIEKVIKDAT >3plus1_Key_669_Nterm (sequence number 27,327) RAVKKLDEIVKEVAKKLEDVVRANEELWRALVELNKESVRRLREIVERVARDLEETAR >3plus1_Key_670_Nterm (sequence number 27,328) SDERRIAERIRELLRESKKLVRDVVEEAKRLLKENRDSTRKIIEDIRRLLRKIEDSTR >3plus1_Key_670_Cterm (sequence number 27,329) SKEETLRKEAEDLLRRLEELTRRLEKKARELLERAKKLSRDLAEELKRLLKELREKGV >3plus1_Key_670_Cterm (sequence number 27,330) AAKRLVEELLKAVTDLSRKNKRILEELLKAIETLSDENKKILTEILDALRRLVEKIEK >3plus1_Key_670_Cterm (sequence number 27,331) KEREEVKEKLDRLLEEVEKTVRELKREHDELLKEVEKLVRDLKKEHDELLKKVKDDGV >3plus1_Key_670_Nterm (sequence number 27,332) DRLDKVEELVKKLLEDTKRTVDRVRELVRKILKKSRETLEELERLIEKILRELEKDAR >3plus1_Key_670_Cterm (sequence number 27,333) SERETVKRRLEELLKEVKRTLDKLKEEHDRLLEDVRRVVEELKREHDKLLKEVKDSGV >3plus1_Key_670_Nterm (sequence number 27,334) SEDEIIKKIIEDLRRVLKEVEEIHKEVEERLDKVLKEAEEMHKEVLKELDRVLDEVKR >3plus1_Key_670_Nterm (sequence number 27,335) SREEVLRELEEVIEDNRRLLEELIEKSKKVLDESLKLIDELLRRLEEVLERVLRLLEE >3plus1_Key_670_Nterm (sequence number 27,336) ISEDDLKRVVDEVEKKLRELKRRYAEALERIKEKIKELKDRYERAVREVVAELRKTTK >3plus1_Key_670_Nterm (sequence number 27,337) SKAEEIAEKLDRLLEENRRALEEITTRLDDLLRRNKDALRKVMEKLKRLLDDLRRGGI >3plus1_Key_671_Cterm (sequence number 27,338) VDSERVKEILERILRVVEEAVRLNEESLRRILDVVRKAVKLDRESLKKILDVVEEAVR >3plus1_Key_671_Cterm (sequence number 27,339) VKDDEVEREIRRVKEDLDRILEEYRRLLEEIKRKLEEILRRVEELHRRLRRKLEEIDR >3plus1_Key_671_Cterm (sequence number 27,340) SRVEELKKLIEDILRISREVVERIKRVAEDIHRINRRVLDDLRKLIEDILRTVEEILA >3plus1_Key_671_Cterm (sequence number 27,341) RGADALSRLLEELLRVVDDLIRVLKELIDKSRKVIEELLELLKRINEENLKVLAEIIK >3plus1_Key_672_Cterm (sequence number 27,342) RGADALSRLLEELLRVVDDLIRVLKELIDKSRKVIEELLELLKRINEENLKVLAEIIK >3plus1_Key_67>3_Nterm (sequence number 27,343) EALRKLVELLVEVLRRLIRVNRELVKLLREVLERLLRILRESVKKLKRLIEKVIKDAT >3plus1_Key_67>3_Cterm (sequence number 27,344) SEKEDAARKLRKLVEELTREYEELVKKLERLIEEIEKVSEESVRKLEKLLAEISEEVR >3plus1_Key_67>3_Nterm (sequence number 27,345) SEKEDAARKLRKLVEELTREYEELVKKLERLIEEIEKVSEESVRKLEKLLAEISEEVR >3plus1_Key_674_Nterm (sequence number 27,346) SEKEDAARKLRKLVEELTREYEELVKKLERLIEEIEKVSEESVRKLEKLLAEISEEVR >3plus1_Key_674_Cterm (sequence number 27,347) RAVKKLDEIVKEVAKKLEDVVRANEELWRALVELNKESVRRLREIVERVARDLEETAR >3plus1_Key_675_Nterm (sequence number 27,348) RAVKKLDEIVKEVAKKLEDVVRANEELWRALVELNKESVRRLREIVERVARDLEETAR >3plus1_Key_676_Nterm (sequence number 27,349) RAVKKLDEIVKEVAKKLEDVVRANEELWRALVELNKESVRRLREIVERVARDLEETAR >3plus1_Key_677_Nterm (sequence number 27,350) SDERRIAERIRELLRESKKLVRDVVEEAKRLLKENRDSTRKIIEDIRRLLRKIEDSTR >3plus1_Key_677_Cterm (sequence number 27,351) SKEETLRKEAEDLLRRLEELTRRLEKKARELLERAKKLSRDLAEELKRLLKELREKGV >3plus1_Key_678_Nterm (sequence number 27,352) SKEETLRKEAEDLLRRLEELTRRLEKKARELLERAKKLSRDLAEELKRLLKELREKGV >3plus1_Key_678_Cterm (sequence number 27,353) SKEETLRKEAEDLLRRLEELTRRLEKKARELLERAKKLSRDLAEELKRLLKELREKGV >3plus1_Key_678_Cterm (sequence number 27,354) ISEDDLKRVVDEVEKKLRELKRRYAEALERIKEKIKELKDRYERAVREVVAELRKTTK >3plus1_Key_678_Nterm (sequence number 27,355) SKAEEIAEKLDRLLEENRRALEEITTRLDDLLRRNKDALRKVMEKLKRLLDDLRRGGI >3plus1_Key_678_Nterm (sequence number 27,356) VDSERVKEILERILRVVEEAVRLNEESLRRILDVVRKAVKLDRESLKKILDVVEEAVR >3plus1_Key_679_Cterm (sequence number 27,357) SKAEEIAEKLDRLLEENRRALEEITTRLDDLLRRNKDALRKVMEKLKRLLDDLRRGGI >3plus1_Key_679_Nterm (sequence number 27,358) SKAEEIAEKLDRLLEENRRALEEITTRLDDLLRRNKDALRKVMEKLKRLLDDLRRGGI

[0051] In certain embodiments, key polypeptides share 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along their length to the amino acid sequences of the key polypeptides in Table 2 (polypeptides having odd-numbered SEQ ID NOs: 27127-27277), Table 3, and / or Table 4. In another embodiment, the key polypeptides share 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along their length with the amino acid sequences of the key polypeptides in Table 3. In another embodiment, the key polypeptides share 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along their length with the amino acid sequences of the key polypeptides in Table 4. In one embodiment of each of the above, the percent specificity may be determined without the optional N- and C-terminal 60 amino acids, and in another embodiment, the percent specificity may be determined with the optional N- and C-terminal 60 amino acids.

[0052] The polypeptides of the present disclosure (i.e., cage polypeptides and key polypeptides) may contain additional residues at the N-terminus, C-terminus, internally, or combinations thereof of the polypeptide, and these additional residues are not included in determining the percent identity of the polypeptide of the present invention compared to the reference polypeptide. Such residues may be any residue suitable for the intended use, including but not limited to tags. As used herein, "tags" include general detectable moieties (i.e., fluorescent proteins, antibody epitope tags, etc.), therapeutic agents, purification tags (such as His tags), linkers, ligands suitable for purification purposes, ligands that facilitate localization of the polypeptide, peptide regions that add functionality to the polypeptide, and the like. Examples are provided herein.

[0053] In one embodiment, the polypeptide is a fusion protein comprising a cage polypeptide disclosed herein fused to a key polypeptide disclosed herein. In one embodiment, the fusion protein comprises a cage polypeptide fused to a key polypeptide, wherein the cage polypeptide is not activated by the key polypeptide. As described herein, the orthogonal LOCKR designs (see FIG. 3) are indicated with lowercase subscripts and are designated LOCKR a Cage a and key a It consists of LOCKR b Cage b and key b This is a cage a is key a Activated only by the cage b is key b Thus, for example, the fusion protein may be activated only by the key b Cage fused to a polypeptide a Such embodiments may include polypeptides. Such embodiments may be used in combination, for example, to improve control of orthogonal LOCKR designs (e.g., LOCKR1 is a cage a -key b The fusion polypeptide comprises LOCKR2, b-key a fusion polypeptide so that they can be expressed in the same cell).

[0054] In one embodiment of the fusion proteins disclosed herein, the cage polypeptide and key polypeptide components of the fusion protein comprise at least one cage polypeptide and at least one key polypeptide having amino acid sequences that have at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along their length, respectively, to cage polypeptides and key polypeptides in different rows of Table 1, Table 2, Table 3, and / or Table 4 (i.e., each cage polypeptide in row 1, column 1 of a table can be fused to any key polypeptide in row 1, column 2, etc.). [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 3

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

[0055] As used throughout this application, the term "polypeptide" is used in its broadest sense and refers to a sequence of subunit amino acids. The polypeptides of the present invention may contain L-amino acids + glycine, D-amino acids + glycine (which are resistant to L-amino acid specific proteases in vivo), or a combination of D- and L-amino acids + glycine. The polypeptides described herein may be chemically synthesized or recombinantly expressed. The polypeptides may be conjugated to other compounds, such as by pegylation, HESylation, PASylation, glycosylation, to promote increased half-life in vivo, or may be produced as Fc fusions or in deimmunized variants. Such conjugations may be covalent or non-covalent, and are understood by those skilled in the art.

[0056] In a fifth aspect, the present disclosure provides a nucleic acid encoding a polypeptide of any embodiment or combination of embodiments of each aspect disclosed herein. The nucleic acid sequence may comprise single-stranded or double-stranded RNA or DNA in genomic or cDNA form, or a DNA-RNA hybrid, each of which may comprise chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such recombinant nucleic acid sequences may comprise additional sequences that are useful for facilitating expression and / or purification of the encoded protein, including, but not limited to, polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings of the present specification, it will be clear to one skilled in the art which nucleic acid sequences encode the fusion proteins of the present disclosure.

[0057] In a sixth aspect, the present disclosure provides an expression vector comprising a nucleic acid of any of the aspects of the present disclosure operably linked to a suitable control sequence. An "expression vector" includes a vector that operably links a nucleic acid coding region or gene to any control sequence that can effect expression of a gene product. A "control sequence" operably linked to a nucleic acid sequence of the present disclosure is a nucleic acid sequence that can effect expression of a nucleic acid molecule. Control sequences need not be contiguous with a nucleic acid sequence, so long as they function to direct its expression. Thus, for example, intervening non-translated but already transcribed sequences can be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including, but not limited to, plasmid and viral-based expression vectors. The control sequences used to drive expression of the disclosed nucleic acids in mammalian systems can be constitutive (driven by any of a variety of promoters including, but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid responsive). The expression vector must be replicable in the host organism, either as an episome or by integration into the host chromosomal DNA. In various embodiments, the expression vector can comprise a plasmid, a viral-based vector, or other suitable expression vector.

[0058] In a seventh aspect, the present disclosure provides a host cell comprising a nucleic acid or expression vector (i.e., episomally or chromosomally integrated) disclosed herein, the host cell can be either prokaryotic or eukaryotic. The cell can be transiently or stably engineered to incorporate an expression vector of the present disclosure, using techniques including, but not limited to, bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome-, DEAE-dextran-, polycation-, or viral-mediated transfection. In one embodiment, the recombinant host cell is (a) a first nucleic acid encoding a polypeptide of any embodiment or combination of embodiments of a cage polypeptide of aspects 1-3 of the present disclosure, operably linked to a first promoter; (b) a second nucleic acid encoding a polypeptide of any embodiment or combination of embodiments of a key polypeptide of aspect 4 of the present disclosure, wherein the key polypeptide binds to a structural region of the cage polypeptide and induces a conformational change in the cage polypeptide, and the second nucleic acid is operably linked to a second promoter.

[0059] The recombinant host cell may contain a single cage polypeptide-encoding nucleic acid and a single key polypeptide-encoding nucleic acid, or may contain multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) first and second nucleic acids, such that in one embodiment, each second nucleic acid may encode a key polypeptide capable of binding to a structural region and inducing a conformational change in a different cage polypeptide encoded by the multiple first nucleic acids. In another embodiment, each second nucleic acid may encode a key polypeptide capable of binding to a structural region and inducing a conformational change in more than one cage polypeptide encoded by the multiple first nucleic acids.

[0060] Thus, in one embodiment, the first nucleic acid comprises a plurality of first nucleic acids encoding a plurality of different cage polypeptides. In one such embodiment, the second nucleic acid comprises a plurality of second nucleic acids encoding a plurality of different key polypeptides, the plurality of different key polypeptides comprising one or more key polypeptides capable of binding to only a subset of the plurality of different cage polypeptides and inducing a conformational change in the subset. In another such embodiment, the second nucleic acid encodes a single key polypeptide capable of binding to each of the different cage polypeptides and inducing a conformational change.

[0061] In another embodiment, the host cell comprises a nucleic acid and / or an expression vector capable of expressing a fusion protein disclosed herein, the host cell comprising: (a) a first nucleic acid encoding a first fusion protein (i.e., a cage polypeptide fused to a key polypeptide) linked to a first promoter; (b) a second nucleic acid encoding a second fusion protein operably linked to a second promoter; (i) a cage polypeptide encoded by a first nucleic acid is activated by a key polypeptide encoded by a second nucleic acid; (ii) the cage polypeptide encoded by the first nucleic acid is not activated by the key polypeptide encoded by the first nucleic acid; (iii) the cage polypeptide encoded by the second nucleic acid is activated by the key polypeptide encoded by the first nucleic acid; (iv) the cage polypeptide encoded by the second nucleic acid is not activated by the key polypeptide encoded by the second nucleic acid.

[0062] In all these embodiments, the first and / or second nucleic acid may be, for example, in the form of an expression vector, hi other embodiments, the first and / or second nucleic acid may be in the form of a nucleic acid integrated into the host cell genome.

[0063] A method for producing a polypeptide according to the present disclosure is an additional part of the present disclosure. In one embodiment, the method comprises: (a) culturing a host according to this aspect of the present disclosure under conditions conducive to expression of the polypeptide; and (b) optionally recovering the expressed polypeptide. The expressed polypeptide can be recovered from a cell-free extract or from the culture medium. In another embodiment, the method comprises chemically synthesizing the polypeptide.

[0064] In an eighth aspect, the present disclosure provides a kit. In one embodiment, the kit comprises: (a) one or more polypeptides (i.e., cage polypeptides) according to any embodiment or combination of embodiments of aspects 1-3 of the present disclosure; (b) one or more polypeptides (i.e., key polypeptides) of any embodiment or combination of embodiments of aspect 4 of the present disclosure; and (c) optionally, one or more fusion proteins of any embodiment disclosed herein.

[0065] In another embodiment, the kit comprises: (a) a first nucleic acid encoding a cage polypeptide of any embodiment or combination of embodiments of aspects 1-3 of the present disclosure; (b) a second nucleic acid encoding a key polypeptide of any embodiment or combination of embodiments of aspect 4 of the present disclosure; and (c) optionally, a third nucleic acid encoding a fusion protein of any embodiment disclosed herein.

[0066] In another embodiment, the kit comprises: (a) a first expression vector comprising a first nucleic acid encoding a cage polypeptide of any embodiment or combination of embodiments of aspects 1-3 of the present disclosure, wherein the first nucleic acid is operably linked to a first promoter; and (b) a second expression vector comprising a second nucleic acid encoding a key polypeptide of any embodiment or combination of embodiments of aspect 4 of the present disclosure, wherein the second nucleic acid is operably linked to a second promoter.

[0067] In each of the kit embodiments, the first nucleic acid, second nucleic acid, first expression vector, and / or second expression vector may comprise a single nucleic acid encoding or expression vector capable of expressing a cage or key polypeptide, or may comprise multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) first nucleic acids, second nucleic acids, first expression vectors, and / or second expression vectors. In various such embodiments, each second nucleic acid may encode, or each second expression vector may be capable of expressing, a key polypeptide capable of binding to a structural region and inducing a conformational change in a different cage polypeptide that is encoded by the multiple first nucleic acids or can be expressed by the first expression vector. In other embodiments, each second nucleic acid may encode, or each second expression vector may be capable of expressing, a key polypeptide capable of binding to a structural region and inducing a conformational change in one or more cage polypeptides that is encoded by the multiple first nucleic acids or can be expressed by the multiple first expression vectors.

[0068] In one embodiment, the promoter operably linked to the nucleic acid encoding the cage polypeptide (first promoter) is different from the promoter operably linked to the nucleic acid encoding the key polypeptide (second promoter) and controls expression of the key polypeptide, thereby allowing for tunable control of the cage polypeptide and any functional polypeptides. In other embodiments, the promoter operably linked to the nucleic acid encoding the cage polypeptide (first promoter) is the same as the promoter operably linked to the nucleic acid encoding the key polypeptide (second promoter). In other embodiments, the first promoter and / or the second promoter can be inducible promoters.

[0069] In a ninth aspect, the present disclosure provides a LOCKR switch, (a) a cage polypeptide comprising a structural domain and a latch domain, the latch domain further comprising one or more biologically active peptides, wherein the structural domain interacts with the latch domain to prevent activity of the biologically active peptide(s); (b) an optional key polypeptide that binds to the cage domain, thereby displacing the latch domain and activating the bioactive peptide(s); (c) optionally, an effector polypeptide(s) that binds to the bioactive peptide(s) when the bioactive peptide(s) are activated.

[0070] The cage polypeptide and its structural and latch regions are discussed above, as are the bioactive peptides, effector polypeptides, and key polypeptides. Any of the embodiments of the cage polypeptides, bioactive peptides, and key polypeptides disclosed herein can be used in the LOCKR switches and kits of the present disclosure. For example, in one embodiment, the cage polypeptide is (a) A helical bundle containing 2 to 7 alpha helices; (b) an amino acid linker connecting each alpha helix. In one embodiment, a key polypeptide is present, which may include a key polypeptide of any embodiment or combination of embodiments disclosed herein.

[0071] In another embodiment, an effector polypeptide is present, the effector polypeptide comprising a polypeptide that selectively binds to the bioactive peptide. Any suitable effector polypeptide can be used depending on the bioactive peptide of interest. In various non-limiting embodiments, the effector peptide can include Bcl2, GFP1-10, proteases, and the like.

[0072] The present disclosure also provides a LOCKR switch comprising a cage polypeptide and a key polypeptide as described herein. In some embodiments, the LOCKR switch comprises (a) a cage polypeptide comprising a structural region and a latch region, the latch region further comprising one or more bioactive peptides, and (b) a key polypeptide that binds to the cage structural region. In some embodiments, the LOCKR switch comprises (a) a cage polypeptide comprising a structural region and a latch region, the latch region further comprising one or more bioactive peptides, and (b) a key polypeptide that binds to the cage structural region, the one or more bioactive peptides in the latch region bind to or interact with one or more effector polypeptide(s). In other embodiments, the LOCKR switch comprises (a) a cage polypeptide comprising a structural region and a latch region, the latch region further comprising one or more bioactive peptides, the structural region interacting with the latch region to prevent activity of the one or more bioactive peptides, and (b) a key polypeptide that binds to the cage structural region, thereby displacing the latch region and activating the one or more bioactive peptides. In some other embodiments, the LOCKR further comprises one or more effector polypeptide(s) that bind to the one or more bioactive peptides when the one or more bioactive peptides are activated.

[0073] In some embodiments, both the latch region and the key polypeptide can bind or interact with the structural region in the corresponding cage polypeptide. The interaction between the latch region and the structural region in the cage polypeptide can be an intramolecular interaction, and the interaction between the key polypeptide and the structural region of the corresponding cage polypeptide can be an intermolecular interaction. However, in some embodiments, the affinity of the latch region for the structural region of the cage polypeptide is higher than the affinity of the key polypeptide for the structural region of the cage polypeptide in the absence of the effector polypeptide.

[0074] In some embodiments, the affinity of the latch region for the structural region of the cage polypeptide is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, or at least about 30-fold greater than the affinity of the key polypeptide for the structural region of the cage polypeptide in the absence of the effector polypeptide. In some embodiments, the affinity of the latch region for the structural region of the cage polypeptide is at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2.0 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.3 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, or at least about 3.0 fold greater than the affinity of the key polypeptide for the structural region of the cage polypeptide in the absence of the effector polypeptide.In some embodiments, the affinity of the latch region for the structural region of the cage polypeptide is at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 110 fold, at least about 120 fold, at least about 130 fold, at least about 140 fold, at least about 150 fold, at least about 160 fold, at least about 170 fold, at least about 180 fold, at least about 190 fold, at least about 200 fold, at least about 210 fold, at least about 220 fold, at least about 230 fold, at least about 240 fold, at least about 250 fold, at least about 260 fold, at least about 270 fold, at least about 280 fold, at least about 290 fold, at least about 300 fold, at least about 310 fold, at least about 320 fold, at least about 330 fold, at least about 340 fold, at least about 350 fold, at least about 360 fold, at least about 370 fold, at least about 380 fold, at least about 390 fold, at least about 400 fold, at least about 410 fold, at least about 420 fold, at least about 430 fold, at least about 440 fold, at least about 450 fold, at least about 460 fold, at least about 470 fold, at least about 480 fold, at least about 490 fold, at least about 500 fold, at least about 510 fold, at least about 520 fold At least about 150 times, at least about 160 times, at least about 170 times, at least about 180 times, at least about 190 times, at least about 200 times, at least about 210 times, at least about 220 times, at least about 230 times, at least about 240 times, at least about 250 times, at least about 260 times, at least about 270 times, at least about 280 times, at least about 290 times, at least about 300 times, for example, about 30 times to about 300 times, for example, about 100 times to about 300 times, about 50 times to about 100 times higher.

[0075] In other embodiments, the intramolecular latch-cage affinity is higher than the intermolecular key-cage affinity, and in the presence of an effector protein, the intermolecular key-cage affinity is higher than the intramolecular latch-cage affinity. As a result, the function of the bioactive peptide depends on the presence of the cage, the key, and the effector protein.

[0076] In certain embodiments, the intermolecular key-cage interaction can overcome the latch-cage interaction in the absence of the effector protein. When the key is absent, the latch-cage affinity is higher than the latch-effector protein affinity (through the binding of the bioactive peptide to the effector protein), and when the key is present, the latch-effector protein affinity (through the binding of the bioactive peptide to the effector protein) is higher than the latch-cage affinity. As a result, the function of the bioactive peptide depends on the presence of the cage, the key, and the effector protein.

[0077] As disclosed herein, a cage polypeptide can be used in conjunction with a key polypeptide by using a cage polypeptide to sequester a bioactive peptide in a latch region, which remains inactive until the key polypeptide displaces the latch by competitive intermolecular binding that induces a conformational change, exposing the encoded bioactive peptide or domain and activating the system (see FIG. 1). The combined use of cage and key polypeptides is described in detail herein in the Examples below and is referred to as a LOCKR switch. LOCKR stands for Latching Orthogonal Cage-Key pRotiens, and each LOCKR design consists of two separate polypeptide chains, a cage polypeptide and a key polypeptide. The orthogonal LOCKR designs (see FIG. 3) are labeled with lowercase subscripts and are designated LOCKR. a Cage a and key a It consists of LOCKR b Cage b and key b This results in a cage a is key a Activated only by the cage b is key b and so on. The prefixes in the polypeptide and LOCKR names indicate the functional groups encoded and controlled by the LOCKR switch. For example, BimLOCKR refers to an engineered switch encoding a Bim peptide, and GFP11-LOCKR refers to an engineered switch encoding GFP11 (the 11th strand of GFP). See Figure 8 for sequence alignments comparing the original LOCKR_a cage scaffold design with its asymmetric (1fix-short-noBim(AYYA)-t0) and orthogonal (LOCKRb~f) designed counterparts.

[0078] In another embodiment, the cage nomenclature is distinguished by 1fix-short and 1fix-latch cages as defined above. aThese show similar but distinct embodiments of a The functional group encoded in the latch is identified by the third part of the name, with a suffix indicating the presence of a toehold. For example, 1fix-short-Bim-t0 encodes Bim in the 1fix-short scaffold without the toehold. In another example, 1fix-latch_Mad1SID_T0_2 indicates that the 1fix-latch scaffold was used to encode Mad1SID without residues. The suffix 2 indicates that there are two versions in which the functional sequence is encoded at different positions in the latch region.

[0079] In one embodiment of the eighth and ninth aspects of the disclosure, the one or more cage polypeptides and the one or more key polypeptides comprise at least one cage polypeptide and at least one key polypeptide in the same row of Tables 1, 2, 3, and / or 4. As will be understood by one of skill in the art based on the teachings herein, such kits can include multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more) cage polypeptide and key polypeptide pairs that can be used together as a LOCKR switch.

[0080] In one embodiment of the kit or switch disclosed herein, the one or more cage polypeptides and the one or more key polypeptides comprise at least one cage polypeptide and at least one key polypeptide having an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along its length to a cage polypeptide and key polypeptide, respectively, in the same row of Tables 1, 2, 3, and / or 4.

[0081] In a tenth aspect, the disclosure provides for the use of the polypeptides, kits, and / or LOCKR switches disclosed herein to sequester bioactive polypeptides in a cage polypeptide, holding them in an inactive ("off") state until combined with a key polypeptide to induce a conformational change that activates ("on") said bioactive peptide. Details of exemplary such uses and methods are disclosed throughout.

[0082] In one embodiment of the kit or switch disclosed herein, the one or more cage polypeptides and the one or more key polypeptides comprise at least one cage polypeptide and at least one key polypeptide having an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along its length to a cage polypeptide and key polypeptide, respectively, in the same row of Tables 1, 2, 3, and / or 4.

[0083] In another embodiment of the eighth and ninth aspects of the present disclosure, the one or more cage polypeptides and the one or more key polypeptides comprise at least one cage polypeptide and at least one key polypeptide that are matched by an identification number in the naming convention used herein. As previously mentioned, the orthogonal LOCKR designs (see FIG. 3) are labeled with lowercase subscripts and the LOCKR a Cage a and key a It consists of LOCKR b Cage b and key b This results in a cage a is key a Activated only by the cage b is key betc. The prefixes in the polypeptide and LOCKR names indicate the functional groups that are encoded and controlled by the LOCKR switch. In one embodiment, all three plus one (3+1) and two plus one (2+1) cage and key polypeptides disclosed herein are matched by an identification number.

[0084] In some embodiments, the cage and key polypeptide names include the prefix 2plus1 or 3plus1, where the first number defines the number of helices in the structural region and the second number defines the number of helices in the latch region. The Nterm or Cterm suffix defines whether the latch in the cage component of the kit contains an N- or C-terminus, respectively, and is indicated in brackets [ ]. The Nterm vs. Cterm and numeric suffixes correspond to the same suffix in the key that it is activated by. For example, cage 2plus1_Cage_Cterm_2406 (SEQ ID NO: 27126) is activated by 2plus2_Key_Cterm_2406 (SEQ ID NO: 27127). EXAMPLES

[0085] overview We are developing a general method to design novel protein switches that can sequester bioactive peptides and / or binding domains and hold them in an inactive ("off") state until combined with a second designed polypeptide, called a key, that induces a conformational change that activates (turns"on") the bioactive peptide or binding domain.

[0086] Nomenclature and construction features that define the LOCKR switch: ●LOCKR stands for Latching Orthogonal Cage-Key proteins, and each LOCKR design consists of two separate polypeptide chains, a cage protein and a key protein. • Cages encode bioactive peptides or binding domains that are sequestered in regions of the cage scaffold called latches. The general strategy is to optimize the position of the encoded peptide or binding domain for maximum burial of the functional residues that need to be sequestered, while simultaneously optimizing for burial of hydrophobic residues and for solvent exposure / compensatory hydrogen bonding of polar residues. • The key displaces the latch by competitive intermolecular binding that induces a conformational change, exposing the encoded bioactive peptide or domain and activating the system (Figure 1). ● Orthogonal LOCKR designs (Figure 3) are marked with lowercase subscripts, and LOCKR a Cage a and key a It consists of LOCKR b Cage b and key b This results in a cage a is key a Activated only by the cage b is key b and so on. The prefixes indicate the functional groups encoded and controlled by the LOCKR switch. For example, BimLOCKR refers to an engineered switch that encodes the Bim peptide, and GFP11-LOCKR refers to an engineered switch that encodes GFP11 (the 11th strand of GFP). ● Toehold: The dynamic range of LOCKR activation by the key can be tuned by truncating the latch length and simultaneously weakening the cage-latch interaction and opening up a cage-exposed region where the key can bind as a "toehold" (Figure 2). LOCKR can also be tuned in a similar manner by designing mutations into the latch that weaken the cage-latch interaction (Figures 1-2, Figure 10). The length of the toehold is included as a suffix in the design name. For example, "-t0" means no toehold and "-t9" means a toehold of 9 residues (i.e., the latch is truncated by 9 residues). • When the term "lock" is used in reference to a single polypeptide chain (not in connection with the LOCKR acronym), it is intended to be synonymous with "cage."

[0087] These designs include the first de novo designed proteins that can undergo conformational switching in response to protein binding. They are modular in that they can encode bioactive peptides of all three types of secondary structure in the inactive conformation: alpha helices, beta strands, and loops, and their responsiveness is tunable and can be tuned over a wide dynamic range by varying the length (length of the cage scaffold and / or latch toehold) and / or by mutating residues at the cage-latch contacts. The designed LOCKR switches can be used to control the activity of a wide range of functional peptides. The ability to harness these biological functions with tight, inducible control is useful, for example, for engineering cells (inducible activation of functions, engineering complex logic operations and circuits), developing sensors, developing inducible protein-based therapeutics, and creating novel biomaterials.

[0088] LOCKR Switch Design We set out to design a novel switchable protein system guided by the following general considerations. First, the free energy adjustment required to achieve maximum dynamic range upon addition of a switch-triggering input is less complex at the same site than at distant sites in a system governed by competition between intermolecular and intramolecular interactions (as is common in allosteric biological systems). Second, a stable protein framework with an extended binding surface available for competing interactions has advantages over frameworks that are ordered only upon binding, since the former are more programmable and less likely to engage in off-target interactions. These features are illustrated by the abstract system shown in Fig. 1a, which undergoes thermodynamically triggered switching between binding-incompetent and binding-competent states. The latch (blue) contains a peptide sequence (orange) that can bind to the target (yellow) unless blocked by intramolecular interactions against the cage (turquoise), while a tighter binding key (magenta) outcompetes the latch, allowing the peptide to bind to the target. The behavior of such a system is governed by binding equilibrium constants in separate sub-reactions (Fig. 1a), K open is the release of the latch from the cage, K LT is the latch binding to the target, and K CK is the binding of the key to the cage. The solution to this set of equations shows that if the latch-cage interaction is too weak (red and orange curves), the system is leaky and the key is less likely to induce folding, whereas if the latch-cage interaction is too strong (purple curve), the system is only partially activated, even at high key concentrations. The latch-cage interaction that yields optimal switching (Fig. 1b, blue curve on the left, green curve on the right) is a function of the latch-target binding affinity. We used this model to guide the design of optimal switchable protein systems, as described in the following sections.

[0089] LOCKR Design Strategy To design such a switchable system, we selected structural features that are amenable to tuning the affinity of the cage-latch and cage-key interactions over a wide dynamic range. Alpha helices have the advantage over beta strands that the inter-helix contacts are dominated by side chain-side chain interactions and therefore more easily tuned than the cooperative backbone hydrogen bonds required for beta sheets. To allow fine control over the relative affinities of the cage-latch and cage-key interactions, we chose to design contacts that involve an embedded hydrogen bond network, allowing for the formation of hydrogen bond donors and acceptors as indicated by Watson-Crick base pairs. 4 , considerable specificity can be obtained by relatively small changes in position 5. As a starting point, the subunit-subunit interaction specificity via hydrogen bond networks (5L6HC3_1) 5 We selected an engineered homotrimer of an α-helical hairpin with the α-helix hairpin α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, α-helix 13, α-helix 14, α-helix 15, α-helix 16, α-helix 17, α-helix 18, α-helix 19, α-helix 19, α-helix 19, α-helix 20, α-helix 21, α-helix 22, α-helix 23, α-helix 24, α-helix 25, α-helix 26, α-helix 27, α-helix 28, α-helix 29 ...9, α-helix 29, α-helix 29, α-helix 29, α-helix 29, α-helix 29, α-helix 29, α

[0090] The 5-helix (cage) and 6-helix (cage and latch) designs were soluble when recombinantly expressed in E. coli, the purified proteins were largely monomeric by size-exclusion chromatography with multi-angle light scattering, and were highly thermostable, remaining folded above 95 °C and in 5 M guanidine hydrochloride (Fig. 1d). Small-angle X-ray scattering (SAXS) spectra were in close agreement with the design model and the previous native trimer (Fig. 1e), suggesting that the structure was not altered by the loop. The 5-helix framework, but not the 6-helix framework, bound to the sixth helix fused to GFP in pull-down assays (Fig. 1f), indicating that the K of the intramolecular interaction is consistent with that of the 6-helix framework when the contacts are otherwise identical. open is reduced by the reduced entropy loss in the formation of intramolecular interactions and its corresponding intermolecular, K CK The latter result is expected since it exceeds open To tune the Latch, we screened destabilizing mutations in the Latch (large hydrophobes for alanine or serine, and alanine residues for large hydrophobes or serine) and used GFP pull-down assays to identify mutants with a range of affinities for the Key. The double mutant, V223S / I238S, binds the Key as strongly as the 5-helix cage without the Latch (Fig. 1e, 10), suggesting that the two serines may weaken the cage-latch interaction because of desolvation losses associated with burying the side chain hydroxyls and because they reduce the helical tendency of the Latch. SAXS and CD spectra show that in the absence of the Key, V223S,I238S is a well-folded 6-helix bundle with a structure similar to the original monomer (Fig. 1d). This Cage-Latch-Key system is termed LOCKR, for Latching Orthogonal Cage-Key pRoteins.

[0091] Regulating Bim-Bcl2 binding and modulating the dynamic range of activation: To attach function to LOCKR, we chose the Bim-Bcl2 interaction central to apoptosis as a model system and attempted to cage Bim such that binding to Bcl2 occurs only in the presence of the key. Constructs with two possible Bim-related sequences engineered onto the latch were engineered Bcl2-binding peptides (aBcl2LOCKR) or only Bim residues important for Bcl2 binding (pBimLOCKR). Each has a different affinity for Bcl2, and in the first set of designs we selected the K LT It is possible to sample a range of values. A Bim-related sequence was grafted onto the latch by sampling different helical registers such that residues involved in binding to Bcl2 were sequestered at the cage-latch contact (data not shown), optimizing burial of hydrophobic residues and surface exposure of polar residues. open can be tuned by non-optimal interactions between the cage and Bim residues or by altering the length of the latch (Fig. 2a). The initial design was tested by biolayer interferometry for binding to Bcl2, showing little Bcl2 binding in the presence or absence of the key. K that is accessible in this system open Value and K CK The range of values ​​is clearly K in this example. LT and the key-induced responses deviated from the ideal curve in Figure 1b.

[0092] We hypothesize that the system can be improved by expanding the contact area presented to the cage, which increases the affinity of the latch to the cage (K open This reduces the number of key insertions required, making it easier to turn the system "off" when the key is not present, while extending the key length allows it to overcome the latch (K open Compared to K CKTaking advantage of the modular nature of the novel parametric helix bundle, the cage, latch, and key were each extended by 5, 9, or 18 residues. To allow the key to outcompete the latch, the latter was extended by K open The LOCKR-binding domain was truncated by 4-9 residues to access a range of binding values, which results in a "toehold" on the cage for binding the KEY. An 18 residue extension with a 9 residue toehold resulted in strongly inducible binding (Figure 2b,c; no Biolayer Interferometry signal was observed with either KEY-bound Bcl2 or with KEY volume added to inactive LOCKR. Activation of binding by the KEY was approximately 40-fold (Figure 2c), comparable to or superior to many naturally occurring processes regulated by protein interactions.

[0093] The key concentration range in which BimLOCKR is activated varies with the length of the key. CK The activation of the key can be controlled by adjusting K since the interaction energy is roughly proportional to the total surface area of ​​the interacting residues. The EC50 for the 58-long designed key is 55.6 + / - 34 nM (Fig. 2c, d) and 230 + / - 58 nM for the 45-residue key. Truncating the additional 5 residues completely abolished activation of the key, indicating that the equilibrium is very sensitive to small changes in the free energy, as expected from the model (Fig. 2d). K LT To examine the function of BimLOCKR across a three-order of magnitude range, we tested its key inducible binding to the Bcl2 homologs BclB and Bak (it binds to Bim with Kds of 0.17 nM (Bcl2), 20 nM (BclB), and 500 nM (Bak)). 6 Biolayer interferometry experiments were performed with immobilized target assayed against the switch with and without the key in solution, and with the key immobilized and assayed alone or together with the target against the switch in solution. From these results, the percentage of target or key binding can be obtained as a function of the concentrations of the switch, key, and target. K open , K CK , and KLT By fitting the model to these data, we obtain open =.01+ / -0.0033, K CK =2.1+ / -1.1nM, K LT (Bcl2) = 28 + / - 7.8 nM, and K LT An estimated value of (BclB) = 32 + / - 22 nM was obtained, and little switch activation was observed, so K LT There are no estimates for (Bak). This fit has an RMSE (root mean square error) of 0.072 nm to the observed BLI data. The approximate agreement of these estimates with the Bim binding Kd (which was not used in the fit) indicates that the thermodynamic model (Figure 1a) is an excellent representation of the system, although it may be missing small properties of the system that affect target binding.

[0094] We next attempted to design a series of orthogonal LOCKR systems with the goal of engineering multiple switches that can be selectively activated in heterogeneous mixtures. Specificity was engineered for use with various hydrogen-bonding networks at the cage-key contacts. The latch helices were then combined with the original extended LOCKR a The new latch / key helix was removed from the model and a backbone for a new sixth helix was created by sampling the radius, helical phase, and z-offset of the new latch / key helix as parameters. The resulting structure was scanned for a new hydrogen-bonding network spanning the contact between the new sixth helix and the cage, along with all buried polar atoms involved in hydrogen bonding, and the remaining contacts around the network were subjected to full sequence and side-chain rotamer optimization using Rosetta™ design. Five designs were selected based on packing quality, sequence dissimilarity, and lack of buried polar atoms not involved in hydrogen bonding. The truncation and toehold mutants were assayed for cognate and off-target key binding using the GFP pull-down assay in Figure 1c. Three of the new designs were found to bind to their cognate keys (Figure 11) and were orthogonal to each other. Keys bound to some extenta All of this is still unknown. The Bim array screws onto the latches of these three designs, and it is the original design, BimLOCKR a (Figure 2) b and BimLOCKR latch c showed 22-fold and 8-fold greater activation, respectively, than their cognate keys that added a 9-residue toehold to the latch (Fig. 3a, b). a , BimLOCKR b , and BimLOCKR c are also orthogonal, with each activated only by its cognate key up to 5 uM concentration (Figure 3c). The effectiveness of the embedded hydrogen bond network approach to achieve specificity is shown by the fact that out of six designed BimLOCKR proteins, three could be successfully switched and activated orthogonally, with a 50% success rate starting from a single scaffold.

[0095] Asymmetric LOCKR switch The original LOCKR switch design (Figures 1-2) was constructed starting from 5L6HC3_1, a newly designed symmetric homotrimer containing six helices. 5 Symmetric repeat motifs create opportunities for misfolding and aggregation. To mitigate these effects, the original LOCKR switch was redesigned to be asymmetric (sequences listed at the end of this document). The asymmetric design exhibited better behavior, was more monomeric, and was experimentally analyzed by X-ray crystal structure (Figure 4) both with the addition of the encoded BIM peptide (Figure 4A) and without the addition of BIM peptide (Figure 4B). The experimental structure without the addition of BIM is nearly identical to the computational design model (Figure 4B), demonstrating the atomic accuracy of the design strategy. Details of the computational design and experimental testing are provided in the Methods section. The base scaffold 5L6HC3_1 used to fabricate LOCKRa (Figure 1) 5 See FIG. 9 for the superposition of the crystal structure of 1fix-short-noBim(AYYA)-t0 (FIG. 4B) with the X-ray crystal structure of (dark).

[0096] gfpLOCKR (GFP11-LOCKR) Using the asymmetric design as a starting point, we successfully encoded the 11th strand of GFP into the designed LOCKR switch (Figure 5). A typical split GFP consists of two parts, strands 1-10 and strand 11, and when mixed, 1-10 binds with 11 to produce fluorescence. Here, we demonstrate that the 11th strand is sequestered in the absence of KEY and cannot bind with GFP-1-10, but readily fluoresces when mixed with KEY in the presence of GFP-1-10 (Figure 5). We experimentally determined the X-ray crystal structure of the designed protein, showing that GFP-11 is structurally encoded as an alpha helix, with a conformation nearly identical to the computer-designed model (Figure 5). This result highlights the efficacy and modularity of the LOCKR system and suggests that it can encode bioactive peptides with secondary structure propensity to be non-helical.

[0097] Colocalization-dependent regulation Figures 1-2 show that the dynamic range of LOCKR activation can be predictively tuned, suggesting that the system can be tuned to respond only when the cage and key are colocalized, which is advantageous for a broad range of functions. Using GFP11-LOCKR in Figure 4, we demonstrated that this is indeed the case and that the engineered LOCKR switch can be tuned to be colocalization-dependent using a Spycatcher™ / Spytag™ fusion (Figure 6). Spycatcher™ is covalently fused to Spytag™, and when the Spycatcher™-fused cage was mixed with a Spytag™-fused key, it showed significantly higher fluorescence than when mixed with a key that was not fused to Spytag™ (Figure 6).

[0098] Caged intein LOCKR switch The designed LOCKR switch, which has a cage component encoding a VMAc intein, shows good activation when mixed with sfGFP and the designed KEY fused to the VMAn intein (Figure 7). SDS-PAGE shows a good VMAc-VMAn reaction, with a band corresponding to the correct molecular weight of the expected spliced ​​protein product (Figure 7).

[0099] Large-scale, high-throughput design of LOCKR switches The original LOCKR switch design (Figures 1-2) was constructed starting from 5L6HC3_1, a newly designed symmetric homotrimer containing six helices. 5 They decided that they should create small LOCKR switches consisting of three or four helices. Using everything they learned from testing and experimental validation of the original LOCKR switch, they created a computational pipeline to automate the design of thousands of new LOCKR switch scaffolds by exhaustively sampling Crick helix parameters. 9 . These 2+1 and 3+1 LOCKR switches have smaller payloads than the original design (advantageous for cell engineering efforts) and, due to their lack of symmetry, may be well-behaved and less prone to aggregation (see the Methods section for details of computational design and experimental testing).

[0100] strepLOCKR (STREPII-LOCKR) Using new 2+1 and 3+1 LOCKR scaffolds from our large-scale high-throughput designs, we designed and tested a novel LOCKR scaffold that encodes and controls the STREPII sequence, (N)WSHPQFEK (sequence number 63) (see Methods section for details). This design (Figure 13A) sequesters the STREPII tag compared to a positive control (Figure 13B) and can be activated in the presence of KEY, as determined by Biolayer Interferometry (Octet) data (Figure 13C-D).

[0101] Data in Figure 12 show caging of the PAH2 domain of the mSin3A transcriptional repressor. See figure legend for details.

[0102] Data in Figure 14 show that the 3 plus 1 LOCKR switch activates GFP fluorescence in response to expression of Key. See figure legend for details.

[0103] Consideration Here we demonstrate the effect of the LOCKR platform by caging a protein-protein interaction inducibly activated by a key. We show in vitro data of caging from binding of Bim peptide to its family members, from GFP strand 11 completing a truncated 1-10 construct, and from binding of anti-StrepTag™ II antibody to caged StrepTag™ II. The modularity and ultrastability of the newly designed proteins allows for tuning of switch activation over a wide dynamic range by adjusting the strength of competing cage-key and cage-latch contacts. Using this method, switches can now be designed beyond these proof-of-concept designs, up to caged peptides for more complex applications. LOCKR is useful for controlling native signaling networks and, in general, for controlling biological functions through fully synthetic networks of novel signaling molecules.

[0104] LOCKR brings the modularity of DNA switching technology to proteins, but with the advantage of being able to control and couple a wide range of biochemical functions carried out by proteins and bioactive peptides, which are far more diverse and extensive than nucleic acids.

[0105] method PCR mutagenesis and isothermal assembly All primers for mutagenesis were obtained from Integrated DNA Technologies (IDT). Mutagenic primers were designed to anneal >18 bp on either side of the site for mutagenesis with the desired mutation encoded in the primer. PCR was used to create fragments with >20 bp overlap upstream and downstream of the mutation site with the desired pET vector. The resulting amplicons were isothermally assembled into either pET21b, pET28b, or pET29b restriction digested with XhoI and NdeI and transformed into chemically competent E. coli XL1-Blue cells. Monoclonal colonies were sequenced by Sanger sequencing. Sequence-verified plasmids were purified using a Qiagen miniprep kit and transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression.

[0106] Synthetic gene construction The synthetic genes were ordered from Genscript Inc. (Piscataway, NJ, USA) and delivered in pET 28b+, pET21b+, or pET29b+ E. coli expression vectors and inserted into the NdeI and XhoI sites of each vector. In the pET28b+ construct, the synthesized DNA was cloned in frame with an N-terminal hexahistidine tag and a thrombin cleavage site, and a stop codon was added to the C-terminus. In the pET21b+ construct, a stop codon was added to the end so that the protein was expressed without the hexahistidine tag. In the pET29b+ construct, the synthesized DNA was cloned in frame with a C-terminal hexahistidine tag. Plasmids were transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression.

[0107] Bacterial protein expression and purification Seed cultures were grown overnight in Lysogeny Broth (LB) or Terrific™ Broth II (TBII) in the presence of 50 μg / mL carbenicillin (pET21b+) or 30 μg / mL (for LB) to 60 μg / mL (for TBII) kanamycin (pET28b+ and pET29b+). Seed cultures were used to inoculate 500 mL Studier TBM-5052 autoinduction medium with antibiotics and grown at 37°C for 24 hours. Cells were harvested by centrifugation at 4000 rcf for 20 minutes at 4°C, resuspended in lysis buffer (20 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0 at room temperature) and lysed by microfluidization in the presence of 1 mM PMSF. Lysates were clarified by centrifugation at 24,000 rcf for at least 30 minutes at 4°C. The supernatant was applied to a Ni-NTA (Qiagen) column pre-equilibrated with lysis buffer. The column was washed twice with 15 column volumes (CV) of wash buffer (20 mM Tris, 300 mM NaCl, 40 mM imidazole, pH 8.0 at room temperature), followed by 15 CV of high salt wash buffer (20 mM Tris, 1 M NaCl, 40 mM imidazole, pH 8.0 at room temperature) and 15 CV of wash buffer. Protein was eluted with 20 mM Tris, 300 mM NaCl, 250 mM imidazole, pH 8.0 at room temperature. Protein was further purified by gel filtration using FPLC and a Superdex™ 75 Increase 10 / 300GL (GE) size exclusion column, and fractions containing monomeric protein were pooled.

[0108] Size Exclusion Chromatography, Multi-Angle Light Scattering (SEC-MALS) SEC-MALS experiments used a Superdex™ 75 Increased 10 / 300GL (GE) size-exclusion column connected to a miniDAWN™ TREOS multi-angle static light scattering and OptilabT-rEX™ (extended range refractometer) detector (Wyatt Technology Corporation, Santa Barbara CA, USA). Protein samples were injected at concentrations of 3–5 mg / mL in TBS (pH 8.0). Data were analyzed using ASTRA™ (Wyatt Technologies) software to estimate the weight-average molar mass (Mw) of the eluted species, as well as the number-average molar mass (Mn) and to assess monodispersity by the polydispersity index (PDI) = Mw / Mn.

[0109] Circular dichroism (CD) measurements CD wavelength scans (260-195 nm) and temperature melts (25-95 C) were measured using an AVIV model 420CD spectrometer. Temperature melts were performed at a heating rate of 4 °C / min, monitoring the absorption signal at 222 nm. Protein samples were 0.3 mg / mL in PBS, pH 7.4 in a 0.1 cm cuvette. Guanidinium chloride (GdmCl) titrations were performed on the same spectrometer with an autotitrator in PBS, pH 7.4 at 25 °C and monitored at 222 nm with a 0.03 mg / mL protein sample in a 1 cm cuvette with a stir bar. Each titration consisted of at least 40 points of uniformly distributed concentration, with a mixing time of 1 min for each step. The titration solution consisted of the same concentration of protein in PBS + GdmCl. GdmCl concentration was determined by refractive index.

[0110] Small-angle X-ray scattering (SAXS) The samples were exchanged into SAXS buffer (20 mM Tris, 150 mM NaCl, 2% glycerol, pH 8.0 at room temperature) by gel filtration. Scattering measurements were performed at the Advanced Light Source on the SIBYLS™ 12.3.1 beamline. The X-ray wavelength (λ) was 1.27 Å, the sample-to-detector distance for the Mar165 detector was 1.5 m, and the scattering range was 0.01–0.59 Å.-1 The scattering vector q (q=4π×sin(θ / λ), 2θ is the scattering angle) range corresponded to a range of 11 keV. The data set was collected using 34 0.2 second exposures over 7 seconds at 11 keV with a protein concentration of 6 mg / mL. Data was also collected at a concentration of 3 mg / mL to determine concentration dependence, and all presented data was collected at higher concentrations where no concentration-dependent aggregation was observed. Data from the 32 exposures were averaged separately in the Gunier, Parod, and Wide-q regions depending on the signal quality across each region and frame. The averages were analyzed using the ScÅtter software package, and data was analyzed and statistics reported. FoXS was used to compare the designed model to the experimental scattering profile and calculate a fit quality (X) value. The hexahistidine tag and thrombin cleavage site on the N-terminus of the LOCKR protein were modeled using Rosetta Remodel™ to ensure that the designed sequence matched the sequence of the experimentally tested protein. To capture the conformational flexibility of these residues, 100 independent models were generated and clustered, and the cluster center of the largest cluster was selected as a representative model for unbiased FoXS fitting.

[0111] GFP pull-down assay His-tagged LOCKR was expressed according to the protocol above from pET28b+, and KEY was expressed fused to superfolder GFP (sfGFP) without the his tag in pET21b+. His-tagged LOCKR was fully purified and dialyzed against TBS (20 mM Tris, 150 mM NaCl, pH 8.0 at room temperature), and KEY-GFP was kept as lysate for this assay. 100 μL of LOCKR above 1 uM was added to a 96-well black Pierce® nickel-coated plate (ThermoFisher) and incubated for 1 hour at room temperature. Samples were discarded from the plate and washed 3 times with 200 μL TBST (TBS + 0.05% Tween-20). 100 μL of lysate containing KEY-GFP was added to each well and incubated for 1 hour at room temperature. Samples were discarded from the plate and washed 3 times with 200 μL TBST (TBS + 0.05% Tween-20). Plates were washed once with TBS and 100 μL of TBS was added to each well. sfGFP fluorescence was measured on a Molecular Devices SpectraMax™ M5 plate reader or a BioTek Synergy Neo2 plate reader. Fluorescence was measured at 485 nm excitation and 530 nm emission with a 20 nm bandwidth for excitation and emission.

[0112] Biolayer Interferometry (BLI) BLI measurements were performed on an Octet® RED96 system (ForteBio) equipped with streptavidin (SA) coated biosensors and all analyses were performed in ForteBio data analysis software version 9.0.0.10. The assay was performed with proteins diluted in HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v / v surfactant P20, 0.5% nonfat dry milk, pH 7.4 at room temperature) from GE. Biotinylated Bcl2 was added to the SA chip up to a threshold of 0.5 nm programmed into the instrument protocol. A baseline was obtained by immersing the loaded biosensor in HBS-EP+ buffer, the association rate was observed by immersing in wells containing a given concentration of LOCKR and KEY, and then the dissociation rate was observed by immersing in the buffer used to obtain the baseline. The rate constants and responses at equilibrium were calculated by fitting a 1:1 binding model.

[0113] Thermodynamic LOCKR model The thermodynamic model in Figure 1a shows three free parameters at five equilibria, which define three equations relating the concentrations of all species (open or closed switch, key, target, switch-key, switch-target, and switch-key-target) at equilibrium. K open =[Switch open ] / [switch closed ] K CK =[Switch open ][key] / [switch-key]=[switch-target][key] / [switch-key-target] K LT =[switch-key][target] / [switch-key-target]=[switch open ][Target] / [Switch-Target]

[0114] The total amount of each component (switch, key, and target) is also constant, constraining the value of each species at equilibrium. This introduces the following equation into the model: [switches]total = [switches open ]+[Switch closed ]+[switch-key]+[switch-target]+[switch-key-target] [key]total=[key]+[switch-key]+[switch-key-target] [Target]Total = [Target] + [Switch-Target] + [Switch-Key-Target]

[0115] These six equations were fed into sympy.nsolve() and a solution was found that yielded six constants (three equilibrium constants, three total concentrations). The binding fractions were extracted from this solution and plotted on the corresponding diagrams.

[0116] Grafting functional sequences onto LOCKR using Rosetta A functional model of LOCKR, created by grafting bioactive sequences into the latch, was designed using Rosetta™ XML with sample grafts starting at every helical register in the latch. The protocol uses two Rosetta movers, SimpleThreadingMover, which changes the amino acid sequence in the latch, and FastRelax™ with default settings to find functional mutations that give the lowest energy structure. Designs were selected by eye in PyMol™ 2.0, and high quality grafts had key binding residues that interacted with the cage and minimized the number of buried unsatisfied hydrogen bond residues.

[0117] Rosetta Design of Orthogonal LOCKR LOCKR aRedesign into an orthogonal cage-key pair using the LOCKR-based ... Hydrogen bond networks were designed using HBNetStapleInterface for selected residues at the contacts. Output included designs with two or three hydrogen bond networks spanning the three helices that make up the contact. All output from HBNet was then designed using PackRotamersMover to place residues at the contacts while maintaining the hydrogen bond networks. Two rounds of design were performed: first using beta_soft to aggressively pack contacts with potentially clashing rotamers while optimizing the interaction energy at the contacts, then using beta to minimize the structure and resolve potentially clashing atoms according to the full Rosetta score function. The final round of design finally optimized interactions across the cage-latch contacts, placing rotamers by the full beta Rosetta score function.

[0118] Candidate orthogonal LOCKR designs were selected based on the lack of unsatisfied buried hydrogen-bonding residues, the number of alanine residues as a proxy for packing quality, and sequence dissimilarity as a metric to find distinct polar / hydrophobic patterns that are sufficient to be orthogonal. Unsatisfied hydrogen-bonding atoms were filtered out using the BuriedUnsatHbonds filter, allowing for the elimination of unsatisfied polar atoms according to the filter criteria. Packing quality was determined by counting alanine residues at the contact points, since a high number of alanines means poorly fitting residues. A maximum of 15 alanine residues were allowed across the three helix contact points. Sequence dissimilarity in all designed pairs of latches was scored by BLOSUM62 by aligning the sequences using Bio.pairwise2 package in BioPython, as shown in seq_alignment.py. The alignment is performed without allowing gaps in the sequences due to the penalty of extensions similar to the structural alignment of two helices, to find the most similar superposition based on large openings and hydrophobic polar patterns. Each score was subtracted from the maximum score to convert the scores to a distance criterion, with the most diverse sequences having the smallest BLOSUM62 scores that converted to the largest distance. The sequences were then clustered using HeirClust_fromRMSD.py with a clustering cutoff of 170, resulting in 13 clusters. The centers of each cluster were selected by maximizing the distance between the 13 selected centers. The 13 candidates were then screened by eye in PyMol™ 2.0 for unsatisfied hydrogen bond atoms and qualitative packing quality. The five best designs by these three metrics were selected using LOCKR. b ~ f It was organized as follows.

[0119] Asymmetric LOCKR switch Original LOCKR Switch awas redesigned by HBNet using Rosetta™, with residues known to be important for LOCKR function remaining fixed, and the remaining residues optimized to maintain hydrophobic packing and introduce sequence diversity that minimizes the number of repeating amino acid sequences and motifs. Synthetic DNA encoding the design was obtained as previously described, and the design was expressed, purified, and biophysically characterized as previously described. Crystallization studies were set up as described in the next section.

[0120] X-ray crystallography Crystallization of protein samples Purified protein samples were concentrated to 12-50 mg / ml in 20 mM Tris, pH 8.0 and 100 mM NaCl. Samples were screened by a 5-position deck Mosquito crystal (ttplabtech) with an active humidity chamber utilizing the crystallization screens JCSG+ (Qiagen), JCSGCoreI-IV (Qiagen), PEG / Ion (Hampton Research), and Morpheus (Molecular Dimensions). The optimal conditions for crystallization of the different designs were as follows: ●1-fix-short-BIM-t0:0.1M Tris, pH8.5, 5% (w / v) PEG8000, 20% (v / v) PEG300, 10% (v / v) glycerol (no need for low temperature) ● 1fix-short-GFP-t0: 0.2M sodium chloride, 0.1M sodium cacodylate, pH 6.5, 2.0M ammonium sulfate (20% glycerol added for low temperature) ● 1fix-short-noBim(AYYA)-t0: 0.2M disodium tartrate, 20% (w / v) PEG3350 (no cold additive)

[0121] X-ray data collection and structure determination Crystals of the designed proteins were looped and added to the corresponding reservoir solution containing 20% ​​(v / v) glycerol in the absence of cryoprotectant and flash frozen in liquid nitrogen. X-ray data sets were acquired at the Advanced Light Source at Lawrence Berkeley National Laboratory by beamlines 8.2.1 and 8.2.2. The data sets were stored in the XDS 35 or HKL2000 36 The initial model was indexed and scaled using either the Phenix™ software suite, using the design model as the initial search model. 38 Program in PHASER(TM) 37 It was created by molecular replacement using Phenix.refine, through refinement by simulated annealing, or, if the resolution is sufficient, by Phenix.autobuild. 40 We worked to reduce model bias using simulated annealing and prime-and-switch phasing with rebuild-in-place set to false using . An iterative process of manual building in COOT and refinement in Phenix™ was used to generate the final models. Due to the high degree of self-similarity inherited in coiled-coil-like proteins, the dataset for reported structures, as reported by Phenix.Xtriage™, complicates structure refinement and may explain the higher than predicted reported R values. RMSDs of bond lengths, angles, and dihedral angles for the idealized geometries were calculated using Phenix™. The overall quality of all final models was assessed using the program MOLPROBITY™.

[0122] gfpLOCKR:(GFP11-LOCKR) switch design and characterization Asymmetric LOCKR aUsing the designed scaffold, the 11th strand of GFP was encoded into a latch as described above in the section "Grafting functional sequences into LOCKR using Rosetta™" and a synthetic gene encoding the designed protein was obtained as described above. The protein was purified and biophysically characterized as described above. To test for induction of fluorescence upon addition of the KEY, the protein was mixed by pipetting and immediately assayed in a black 96-well plate using a Biotek Synergy Neo2 plate reader to monitor relative GFP fluorescence (Ex: 488, Em: 508, 10 min to read). Cage leakage was assessed by measuring GFP fluorescence over time in the absence of the KEY.

[0123] Colocalization-dependent switching by gfpLOCKR (GFP11-LOCKR) in vitro GfpLOCKR cage was cloned with SpyCatcher™ fused to its N-terminus via a floppy linker, gfpLOCKR key was cloned with SpyTag™ fused to its C-terminus via a floppy linker, and GFP1-10 was cloned into its own pET21 vector. These proteins were expressed in E. coli Lemo21 cells overnight at 18°C ​​using Studier's autoinduction medium. After expression, producer cells were harvested by centrifugation and lysed by microfluidizer. The desired protein was purified from the clarified lysate by Ni-NTA affinity chromatography and purified by Nanodrop A. 280Proteins were diluted to final concentrations in PBS (Cage: 1.5uM, 0.8uM, 0.4uM, 0.2uM, 0.094uM, Key: 1.5uM, 0.8uM, 0.4uM, 0.2uM, 0.094uM in all samples; GFP1-10: 1.9uM) and pooled with SpyCatcher™-Cage alone (no key), SpyCatcher™-Cage with naked key (no SpyTag™), and SpyCatcher-Cage with SpyTag-Key. Proteins were mixed by pipetting and immediately assayed in black 96-well plates using a Biotek Synergy Neo2 plate reader to monitor relative GFP fluorescence (Ex: 488, Em: 508, 10 min to read). Cage leakage was assessed by measuring GFP fluorescence over time in the absence of key. Colocalization dependence was confirmed by showing that SpyTag™-Keys activated GFP fluorescence faster than naked Keys.

[0124] Caged intein LOCKR switch The VMAC intein sequence is LOCKR a The VMAn intein sequence was designed to be encoded in the latch of a The intein was fused to the LOCKR construct. The construct was cloned and purified as in the previous LOCKR design above. Intein activity (splicing) was assessed by SDS-PAGE.

[0125] Large-scale, high-throughput design of LOCKR switches In a computational pipeline for designing thousands of new LOCKR switch scaffolds from scratch, the backbones were exhaustively sampled using Crick helix parameters for 3-helix bundles (denoted 2 plus 1 or 2+1 for 2-helix scaffolds and 1-helix latches) and 4-helix bundles (denoted 3 plus 1 or 3+1 for 3-helix and 1-helix latches). Sampled parameters included z-offsets (-1.51, 0, and 1.51), helix phases in 10 degree increments between 0 and 100, and superhelical radii at each helix ranging from 5 to 10 Å from the central superhelical axis (z-axis). Based on the success of the original LOCKR design, we tailored the designs with straight helices and no supercoiling (superhelical twist fixed at 0.0). Each helix created was 58 residues in length, and the Rosetta loop closure method was used to add loops connecting all the helices into a single polypeptide chain (cage scaffold). Sequence and side chain design was performed using HBNet, MC-HBNet, and RosettaDesign™. Additional designs were created by truncating the helix bundle to shorter scaffolds, creating versions with the latch as the N- or C-terminal helix, and by trying different toehold lengths (truncation of the latch helix that ends in a polar residue and removes at least one or two hydrophobic packing residues from the original design).Designs were selected based on computational methods learned from previous iterative testing and design of LOCKR scaffolds and HBNet helix bundles; important criteria included secondary structure shape complementarity (ss_sc) >0.65 (best designs had ss_sc >0.7), a RosettaHoles™ filter on the region surrounding the hydrogen bond network to eliminate designs with large holes adjacent to the hydrogen bond network in the core of the scaffold, designs required to have at least two distinct hydrogen bond networks spanning all helices in the design model (i.e., each helix must contribute at least one amino acid side chain to the network), and the number of Ile, Leu, and Val residues and the number of contacts made by these amino acid types compared to Ala (a smaller amino acid), which correlates well with designs with tight, interdigitated hydrophobic packing and also serves as a surrogate that is important for stable protein scaffolds.

[0126] strepLOCKR (STREPII-LOCKR) computer design: A LOCKR switch encoding the STREPII tag, (N)WSHPQFEK (SEQ ID NO: 63), was designed using 2+1 and 3+1 switches from a large-scale high-throughput LOCKR design set. This sequence is difficult to encode due to Pro (which twists the alpha helix) and Trp and His, which may be involved in hydrogen bonding if buried. To address these issues, rather than sampling every helical residue, we mined a large design set to find LOCKR scaffolds that already contained Trp (W), His (H) preorganized into the hydrogen bond network of the design. Designs with Phe (F) preorganized were also considered.

[0127] strepLOCKR (STREPII-LOCKR) Experimental Trials: Purified proteins were tested for their ability to sequester STREPII sequences in the absence of KEY and activate them in the presence of KEY using biolayer interferometry (Octet® RED96 system, PALL ForteBio), where the THE™ NWSHPQFEK (SEQ ID NO: 63) tag antibody (mAb mouse, Genscript A01732-200) was added to an anti-mouse IgG Fc Capture (AMC) biosensor (PALL ForteBio) and the chip was preconditioned by cycling between glycine, pH 1.65, and Octet assay buffer, GE's HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v / v surfactant P20, 0.5% nonfat dry milk, pH 7.4 at room temperature). Protein samples were diluted in Octet assay buffer, keeping the dilution factor constant to minimize noise. Antibody loading tips were reused up to eight times using the recommended regeneration protocol of cycling between glycine, pH 1.65, and Octet assay buffer (minimal loss of loading was observed when preconditioning the tip and a signal threshold was set to ensure consistent loading of the tip each time).

[0128] THE™ NWSHPQFEK (SEQ ID NO:63) Tag Antibody (mAb Mouse, GenscriptA01732-200) was used at a concentration of 5ug / mL in Octet Assay Buffer, antibody stocks were made up to 0.5mg / mL in 400ul Milli-Q treated HO and stored in aliquots at -80C and thawed immediately before use.

[0129] Purification of proteins from bacterial preparations not previously described: Seed cultures were grown either overnight in Luria-Bertani (LB) medium or for 8 hours in Terrific broth at 37°C in the presence of 50 μg / ml carbenicillin (pET21-NESG) or 50 μg / ml kanamycin (pET-28b+). Seed cultures were used to inoculate 500 mL of LB (induced with 0.2 mM IPTG at OD600 ~0.6-0.9) or Studier autoinduction medium with antibiotics. Cultures were expressed overnight at 18°C ​​(many designs were later expressed at 37°C for 4 hours with no noticeable difference in yield). Cells were harvested by centrifugation at 5000 rcf for 15 min at 4°C, resuspended in lysis buffer (20 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0 at room temperature), and then lysed by sonication in the presence of lysozyme, DNAse, and EDTA-free cocktail protease inhibitors (Roche) or 1 mM PMSF. Lysates were clarified by centrifugation at 18,000 rpm at 4°C for at least 30 min and applied to a Ni-NTA (Qiagen) column pre-equilibrated with lysis buffer. The column was washed three times with 5 column volumes (CV) of wash buffer (20 mM Tris, 300 mM NaCl, 40 mM imidazole, pH 8.0 at room temperature), followed by 3–5 CV of high salt wash buffer (20 mM Tris, 1 M NaCl, 40 mM imidazole, pH 8.0 at room temperature) and 5 CV of wash buffer. Proteins were eluted with 20 mM Tris, 300 mM NaCl, 250 mM imidazole, pH 8.0 at room temperature. No reducing agent was added as none of the designed proteins contained cysteines. References 1. Huang, P.-S., Boyken, SE & Baker, D. The coming of age of de novo protein design. Nature 537, 320-327 (2016). 2.Joh,NHet al.De novo design of a transmembrane Zn 2+-transporting four-helix bundle.Science 346,1520-1524(2014). 3.Davey,J.A.,Damry,A.M.,Goto,N.K.& Chica,R.A.Rational design of proteins that exchange on functional timescales.Nat.Chem.Biol.13,1280-1285(2017). 4.Huang,P.-S.et al.High thermodynamic stability of parametrically designed helical bundles.Science 346,481-485(2014). 5.Boyken,S.E.et al.De novo design of protein homo-oligomers with modular hydrogen-bond network-mediated specificity.Science 352,680-687(2016). 6.Berger,S.et al.Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer.Elife 5,1422(2016). 7.Leaver-Fay,A.et al.ROSETTA3:an object-oriented software suite for the simulation and design of macromolecules.Meth Enzymol 487,545-574(2011). 8.Kuhlman,B.& Baker,D.Native protein sequences are close to optimal for their structures.Proc Natl Acad Sci USA 97,10383-10388(2000). 9.Crick,F.H.C.The packing of[alpha]-helices:simple coiled-coils.Acta Cryst(1953).Q6,689-697[doi:10.1107 / S0365110X53001964]6,1-9(1953). 10.Glantz,S.T.et al.Functional and topological diversity of LOV domain photoreceptors.Proc Natl Acad Sci USA 113,E1442-51(2016). 11.Dueber,J.E.,Mirsky,E.A.& Lim,W.A.Engineering synthetic signaling proteins with ultrasensitive input / output control.25,660-662(2007). 12.Dueber,J.E.,Yeh,B.J.,Chak,K.& Lim,W.A.Reprogramming control of an allosteric signaling switch through modular recombination.Science 301,1904-1908(2003). 13.Huang,P.-S.et al.RosettaRemodel:A Generalized Framework for Flexible Backbone Protein Design.PLoS ONE 6,e24109(2011). 14.Schneidman-Duhovny,D.,Hammel,M.& Sali,A.FoXS:a web server for rapid computation and fitting of SAXS profiles.Nucleic Acids Res 38,W540-4(2010). 15.Maguire,J.B.,Boyken,S.E.,Baker,D.& Kuhlman,B.Rapid Sampling of Hydrogen Bond Networks for Computational Protein Design.J.Chem.Theory Comput 14,2751-2760(2018).

Claims

1. A LOCKR switch, (a) a cage polypeptide comprising a helical bundle comprising 2 to 7 alpha-helices, said cage polypeptide comprising a structural region, a latch region, and a linker, said structural region comprising at least one alpha-helix, said latch region comprising at least one alpha-helix, and said linker connecting said structural region and said latch region; (b) a junction comprising an intramolecular interaction between the structural region and the latch region in the absence of a key polypeptide, wherein the latch region comprises (i) one or more mutations at the junction that destabilize the intramolecular interaction, or (ii) a truncation that destabilizes the intramolecular interaction or exposes the junction or a portion thereof to a key polypeptide; (c) a key polypeptide comprising at least one alpha-helix, the at least one alpha-helix being capable of binding to the contact point through an intermolecular interaction; Including, the affinity of the intermolecular interaction is stronger than the affinity of the intramolecular interaction, in the presence of the key polypeptide, the key polypeptide competes with the latch region for binding to the structural region; In the presence of said key polypeptide, said key polypeptide replaces said latch region at said junction. LOCKR switch.

2. The LOCKR switch described in claim 1, wherein the structural region includes two to six alpha-helices and linkers connecting each alpha-helix to the other alpha-helices.

3. A LOCKR switch as described in claim 1 or 2, wherein when the one or more mutations in the latch region are absent, the sequence of the structural region and the sequence of the latch region at the contact are identical.

4. A LOCKR switch described in any one of claims 1 to 3, wherein when the truncation in the latch region is absent, the sequence of the cage polypeptide and the sequence of the latch region at the contact are identical.

5. A LOCKR switch described in any one of claims 1 to 4, wherein the latch region contains one or more biologically active peptides.

6. A LOCKR switch described in any one of claims 1 to 5, wherein replacement of the latch region exposes the one or more biologically active peptides.

7. A LOCKR switch according to any one of claims 1 to 6, wherein in the presence of a complex formed by the key polypeptide binding to the cage polypeptide, in the presence of an effector polypeptide, the one or more biologically active peptides bind to the effector polypeptide.

8. A LOCKR switch described in any one of claims 1 to 7, wherein the sequence of the key polypeptide and the sequence of the structural region at the contact point are identical.

9. A LOCKR switch described in any one of claims 1 to 8, wherein the contact point comprises an intramolecular interaction and the key polypeptide comprises one or more mutations that destabilize the intramolecular interaction.

10. A LOCKR switch described in any one of claims 1 to 9, wherein the one or more mutations change the position of a hydrogen bond donor or hydrogen bond acceptor.

11. A LOCKR switch described in any one of claims 1 to 10, wherein the one or more mutations include (i) embedding or removal of a hydrophobic residue, and / or (ii) exposing or inserting a polar residue.

12. A LOCKR switch described in any one of claims 1 to 11, wherein the one or more mutations include one or more substitutions of one or more isoleucine (I) residues, leucine (L) residues, and valine (V) residues with alanine (A) residues or serine (S) residues.

13. A LOCKR switch described in any one of claims 1 to 10, wherein the truncation of the latch region includes (i) embedding or removing hydrophobic residues, and / or (ii) exposing polar residues.

14. A LOCKR switch according to any one of claims 1 to 13, wherein the structural region, the latch region, or at least one alpha-helix of the key polypeptide contain the same number of residues.

15. A LOCKR switch described in any one of claims 1 to 14, wherein the at least one alpha-helix of the structural region and the at least one alpha-helix of the latch region contain more residues than the at least one alpha-helix of the key polypeptide, thereby increasing the intramolecular interactions compared to the intermolecular interactions.

16. A LOCKR switch described in any one of claims 1 to 15, wherein the at least one alpha-helix of the structural region and the at least one alpha-helix of the key polypeptide contain more residues than the at least one alpha-helix of the latch region, thereby increasing the intermolecular interactions compared to the intramolecular interactions.

17. A LOCKR switch described in any one of claims 1 to 16, wherein the structural region, the latch region, and at least one alpha-helix of the key polypeptide each independently comprise a length of 18 to 60 amino acids.

18. A LOCKR switch described in any one of claims 1 to 17, wherein the linker comprises 3 to 10 amino acids in length.

19. A LOCKR switch described in any one of claims 1 to 18, wherein the linker comprises a secondary structure, and the secondary structure comprises a loop.

20. A LOCKR switch described in any one of claims 1 to 19, wherein the latch or the one or more biologically active peptides comprise a secondary structure, and the secondary structure comprises a non-helical structure.

21. A LOCKR switch described in any one of claims 1 to 20, wherein the contact contacts the N-terminus of at least one alpha-helix of the latch region.

22. A LOCKR switch described in any one of claims 1 to 20, wherein the contact contacts the C-terminus of at least one alpha-helix of the latch region.

23. A LOCKR switch as described in any one of claims 1 to 22, wherein at least one alpha-helix of the latch region does not include the one or more biologically active peptides at the contact point.

24. A nucleic acid comprising a cage polypeptide or key polypeptide described in any one of claims 1 to 23.

25. A nucleic acid comprising a cage polypeptide and a key polypeptide described in any one of claims 1 to 23.

26. A vector comprising the nucleic acid described in claim 24 or 25.

27. ​​A cell comprising a LOCKR switch described in any one of claims 1 to 23, a nucleic acid described in claim 24 or 25, or a vector described in claim 26.

28. Use of one or more of the LOCKR switch of any one of claims 5 to 23, the nucleic acid of claim 24 or 25, the vector of claim 26, and the cell of claim 27 to sequester one or more bioactive peptides in the cage polypeptide and maintain the bioactive peptides in an inactive state until they combine with the key polypeptide to induce a conformational change that activates the bioactive peptide.