Peptides and method of use in preparing nanodiscs

Abstract

The present disclosure provides a peptide comprising an amino acid sequence of formula (I) (DWX1KAFYDKX2AEKX3KEAX4 (SEQ ID NO: 41)) wherein X1 is an amino acid with hydrophobic side chain; X2 is selected from V or W; X3 is selected from L or W; X4 is selected from F or W; and the N-terminus of the peptide is modified with a fatty acid. Methods of using the peptide to prepare a nanodisc also is provided.

Description

PEPTIDES AND METHOD OF USE IN PREPARING NANODISCSFIELD

[0001] The disclosure relates to peptides and / or nanodiscs and methods of making and using thereof for extraction of proteins.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 617,488, filed on January 4, 2024, the contents of which is hereby incorporated in its entirety by reference.INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIAL

[0003] This application contains, as a separate part of the disclosure, a Sequence Listing in computer-readable form which is incorporated by reference in its entirety and identified as follows: 59555P_SeqListing.XML; Size: 56,158 bytes; Created: December 6, 2023.GOVERNMENT SUPPORT CLAUSE

[0004] This invention was made with government support under grant number GM 140920 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0005] Nanodisc technology allows for in-depth biophysical characterizations of cell membrane complexes that is usually challenging in traditional membrane mimetic systems. The technology also provides a promising platform for cargo delivery to host cells. However, successful reconstitution of membrane proteins in nanodiscs is often an arduous task and requires extensive efforts to identify compatible components and assembly conditions which produce stable nanodiscs with membrane proteins in a substantially native conformation. In addition, the native membrane lipid environment is marked by unique properties that are difficult to recapture with current nanodiscs. Despite the tremendous progress in the development and application of nanodisc technology, there remains significant challenges associated with production of nanodiscs comprising, e.g., membrane protein complexes.SUMMARY

[0006] The present disclosure provides a peptide comprising an amino acid sequence of formula I:DWX1KAFYDKX2AEKX3KEAX4(I) (SEQ ID NO: 41 ) wherein Xi is an amino acid with hydrophobic side chain; X2is selected from V or W; X3 is selected from L or W; X4is selected from F or W; and wherein the N-terminus of the peptide is modified with a fatty acid, such as hexanoic acid.

[0007] Optionally, the peptide the peptide of the present disclosure comprises an amino acid sequence of formula II:DWXiKAFYDKX2AEKX3KEAX4X5W (II) (SEQ ID NO: 42) wherein X5 is selected from D or E.

[0008] In various aspects, the peptide of the disclosure docs not comprise SEQ ID NO: 1. In various aspects, Xi is F, L, or W. In various aspects, Xi is L, X2is V, X3 is W, X4is F, and X5 is D.

[0009] Optionally, the peptide of the present disclosure comprises an amino acid sequence of formula III:DWXIKAFYDKX2AEKX3KEAX4X5WX6K (III) (SEQ ID NO: 43) wherein X& is selected from L or F.

[0010] In various aspects, the peptide of the disclosure is amidated at the C-terminus. In various aspects, the peptide of the disclosure comprises the amino acid sequence of any one of SEQ ID NOs: 22-40.

[0011] The disclosure also provides a method of preparing a nanodisc, the method comprising (a) contacting a lipid bilayer comprising a payload with a peptide comprising an amino acid sequence of formula I:DWXIKAFYDKX2AEKX3KEAX4(I) (SEQ ID NO: 41) wherein the N-terminus of the peptide is modified with a fatty acid; Xi is an amino acid with hydrophobic side chain; X2is selected from V or W; X3 is selected from L or W; X4is selected from F or W; and wherein the N-terminus of the peptide comprises a hexanoic acid fatty acid modification; and (b) purifying a nanodisc comprising the payload, wherein the method does not comprise a reconstitution step using detergent.

[0012] Optionally, the method of the disclosure comprises the peptide comprising an amino acid sequence of formula II:DWX1KAFYDKX2AEKX3KEAX4X5W (II) (SEQ ID NO: 42) wherein X5 is selected from D or E.

[0013] In various aspects, the method of the disclosure comprises a peptide that does not comprise SEQ ID NO: 1. In various aspects, the fatty acid is hexanoic acid. In various aspects, the fatty acid comprises a myristoyl moiety, decanoic acid, or palmitoyl moiety. Optionally, the peptide is amidated at the C-terminus. In various aspects, the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 22-40. In various aspects, the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 7-21.

[0014] In some aspects of the disclosure, the lipid bilayer is a cell membrane fraction and / or the pay load of interest is a protein, such as a transporter protein, a receptor, or an ion channel. In various aspects, the payload of interest is a detergent sensitive protein. In various aspects, the lipid bilayer is an asymmetric membrane and the nanodisc retains membrane asymmetry.

[0015] Additional embodiments and aspects of the presently disclosed compositions and methods are provided below. All headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGs. 1A-1E illustrate nanodiscs and data relating to use of membrane scaffold peptides for detergent-free reconstitution of nanodiscs. FIG. 1A is an illustration of the experimental design to extract a bacterial prototype ATP-binding cassette transporter MalFGK.2 into nanodiscs from proteoliposomes. FIG. IB shows native gel analysis of detergent-free nanodisc reconstitution using the peptides 18A, 22A, and 4F. FIG. 1C shows size-exclusion chromatography (SEC) profiles of MalFGKi detergent- free nanodiscs formed using peptide 18 A, in comparison with control experiments of proteoliposomes (pL). FIG. ID shows negative stain electron microscopy (EM) micrograph of MalFGK.2 nanodiscs encased by Hex-18A. FIG. IE is a graph illustrating ATPase activities (hydrolysis nmol / min / mg) of MalFGK2 in detergentmicelles (DDM), proteoliposomes (pL), and I SA-enclosed nanodiscs (ND) in the presence and absence of maltose.

[0017] FIGs. 2A-2D relate to fatty acid modifications for enhancing the efficacy of detergent- free nanodiscs through amphipathic membrane scaffold peptides (henceforth, “DeFrMSPs”). FIG. 2A shows a native gel analysis of detergent-free nanodisc reconstitution using the indicated peptides (NSP, Hexl8A, Pall8A, and Hex22A). FIG. 2B is a graph illustrating size-exclusion chromatography (SEC) profiles of MalFGKi detergent- free nanodiscs formed using Hex- 18 A. FIG. 2C shows a negative stain electron microscopy (EM) micrograph of MalFGIG nanodiscs encased by Hex- 18 A. FIG. 2D shows single-particle cryo-EM analysis of MalFGK in Hex-18A nanodiscs. Left panel of FIG. 2D shows 2D class average; right illustration of FIG. 2D is a 3D reconstructed model.

[0018] FIGs. 3A-3D illustrate DeFrMSPs enclosing stable and functional membranes and data relating thereto. FIG. 3A is an illustration of the membrane fusion assay described in the Examples. FIG. 3B summarizes fusion activities of DeFrMSP nanodiscs. Abbreviations: protein free (pf), cognate vesicle (v) SNARE, and targeted membrane (t) SNARE. FIG. 3C provides representative results of the sytl -lipid bending assay described in the Examples. PC: l,2-dioleoyl-sn-glycero-3-phosphocholine; PS: l,2-dioleoyl-sn-glycero-3-phospho-l-serine. FIG. 3D depicts a schematic and a representative native gel of an asymmetric membrane trapped in DeFrMSP nanodiscs as demonstrated by SecA-lipid interactions using native electrophoresis.

[0019] FIGs. 4A-4C illustrate the formation of native nanodiscs from bacterial membranes using DeFrMSPs. FIG. 4A (left panel) depicts an illustration of the reconstitution procedure described herein. FIG. 4A (right panel) shows isolation efficiencies of MalFGKi into nanodiscs with different designs of DeFrMSPs. DDM and buffer were used as positive and negative controls for data normalization. FIG. 4B shows negative stain electron microscopy (EM) micrograph of MalFGKj nanodiscs encased by Hex-2OB1WA. Scale bar = 50 nm. FIG. 4C shows representative gels of extracted MalFGKi.

[0020] FIGs. 5A-5C illustrate the reconstitution and characterization of mGluR7 in native nanodiscs. FIG. 5 A is an illustration of mGluR7 interaction with Efn during neuronal signaling. FIG. 5B shows a representative native gel of mGluR7 in Hex-2OB1WA nanodiscs binding toEfn-1 . FIG. 5C shows quantification of mGluR7 interaction with Efn from native gel analysis. Relative binding (%) is provided on the y-axis, Efn concentration is provided on the x-axis.

[0021] FIGs. 6A-6B illustrate the reconstitution and characterization of HCN 1 in native nanodiscs. FIG. 6A is an illustration of the smFRET assay to probe cAMP-triggered conformational changes of HCN1. FIG. 6B is a representative fluorescence-time trace overlaid with idealized fit showing fcAMP binding to HCN1 reconstituted into native nanodiscs at lOOnM concentration.

[0022] FIGs. 7A-7D show the screen of potential candidate scaffolds for native nanodisc reconstitution. FIG. 7A shows a native electrophoresis of nanodiscs formed with the indicated peptides. FIG. 7B shows size exclusion chromatography analysis of nanodiscs and associated lipids. FIG. 7C shows native electrophoresis characterization of nanodiscs formed by polymers. FIG. 7D shows ATPase activities of MalFGKi isolated in polymer-based native nanodiscs. The results show that MalFGK is not functional in these polymer nanodiscs.

[0023] FIGs. 8A-8C show optimization of DeFrMSPs for the formation of native nanodiscs. FIG. 8A depicts native gel analysis of native nanodiscs formed by the indicated DeFrMSPs. FIG. 8B depicts ATPase activities of Hex-18A nanodiscs harboring MalFGKi. FIG. 8C depicts the quantification of lipids in the indicated nanodiscs.

[0024] FIGs. 9A-9D show the reconstitution of native nanodiscs from bacterial membranes. FIG. 9A shows SDS-PAGE analysis of the extraction of MalFGKi from bacterial membrane using the indicated DeFrMSPs in comparison with DDM or negative control. The abbreviations for the wells are as follows: SM, extracted starting materials; E, eluted fraction after affinity purification; FT, flow through fractions; and P, non-solubilized pellet fractions. FIG. 9B shows nanodisc (ND) formation efficiencies with the indicated DeFrMSPs. Data were normalized to Hex-18A. FIG. 9C shows a lipidomic analysis which validates that native lipids were incorporated into native nanodiscs. FIG. 9D shows ATPase measurements of the indicated MalFGKs mutants isolated in native nanodiscs.

[0025] FIGs. 10A-10B show the optimization of extracting mGluR7 into native nanodiscs from HEK293 cell membranes. FIG. 10A shows nanodisc (ND) formation efficiencies with the indicated DeFrMSPs. Data were normalized to the amount of extracted proteins using DDM. FIG. 10B shows an SDS-PAGE analysis of extracted mGluR7 at the indicated conditions.Labels of the wells are as follows: 1 , Hex-18A; 2, Hex20B; 3, Hex20BlWl ; and 4, Hcx20BF4W.

[0026] FIGs. 11A-11B show the optimization of extracting HCN1 into native nanodiscs from HEK293 cell membranes. FIG. 11A shows nanodisc (ND) formation efficiencies with the indicated DeFrMSPs. Data were normalized to the amount of extracted proteins using DDM. FIG. 1 IB shows a TLC analysis of the indicated native nanodiscs.

[0027] FIGs. 12A-12F depict the characterization of MalGFK2 nanodiscs extracted from proteoliposomes using peptide and polymer scaffolds. FIG. 12A depicts a representative image of an electrophoresis gel of detergent-free nanodisc formation by peptide scaffolds. MalFGK.2 proteoliposomes were incubated with increasing concentrations (33, 100, and 300 pM) of the indicated scaffold peptides or DDM (0.5%) and analyzed by blue native electrophoresis. FIG. 12B depicts a graph of lipid quantification in MalFGK NDs formed with the indicated peptide scaffolds. FIG. 12C depicts a representative image of an electrophoresis gel of detergent free ND formation by polymer scaffolds. MalFGK2 proteoliposomes were incubated with increasing concentrations (0.5% and 1.5%) of the indicated scaffold polymers and analyzed by blue native electrophoresis. FIG. 12D depicts a graph of ATPase activities of polymer-encased MalFGK.2 NDs. Data are shown as mean ± s.d., n = 3 independent experiments. FIGs. 12E-12F depict the aggregation of polymer NDs in the presence of MgCh. FIG. 12E depicts a representative image of an electrophoresis gel of sample aggregation of MalFGK2 NDs formed with the indicated polymers or scaffold peptides were treated with or without MgCh at room temperature for 10 mins. FIG. 12F depicts a graph of the data of FIG. 12E.

[0028] FIGs. 13A-13D depict the optimization and characterization of DeFrMSPs for detergent-free formation of MalFGKh NDs. FIG. 13A depicts a representative image of an electrophoresis gel of MalFGK2 proteoliposomes were incubated with increasing concentrations (33 and 100 pM) of the indicated scaffold peptides or DDM (0.5%) and analyzed by blue native electrophoresis to screen DeFrMSP designs to potentiate the performance in extracting MalFGK.2 from proteoliposomes into NDs. FIG. 13B depicts a graph of the ATPase activities of MalFGK2 NDs formed with Hex-18A. Data are shown as mean ± s.d., n = 3 independent experiments. FIG. 13C depicts a graph of the measurement of lipids extracted into MalFGKj NDs by Hex- 18A. MalFGK.2 proteoliposomes were prepared with lipid mixtures harboring 0.5% Rho-PE andconverted into NDs by Hex-18A. Samples were characterized by SEC using a Superdex200 3.2 / 300 column. FIG. 13D depicts a graph of the diameters of MalFGI NDs determined from negative stain EM in FIG. 13C.

[0029] FIGs. 14A-14D depict the CryoEM analysis of MalFGK2 NDs. FIG. 14A depicts a schematic of the overall workflow for data processing. FIG. 14B depicts a graph of Fourier Shell Correlation (FSC) curves for the overall resolution. FIG. 14C depicts a graph of the Map-to- model fit between the full map and PDB coordinates. FIG. 14D depicts a schematic of the local resolution.

[0030] FIGs. 15A-15G depict the characterization of protein-free DeFrNDs. FIGs. 15A-15F depict graphs of different lipid mixtures that were incubated with DeFrMSP (Hex 18 A) at the indicated peptide / lipid ratios (4°C, O / N) and characterized by SEC on a Superose 6 10 / 300 column equilibrated in Buffer A and calibrated using circularized NDs with defined diameters. FIG. 15G depicts a graph demonstrating that lipid exchange between DeFrNDs was much lower than polymer NDs. Empty NDs (0.5 pM) harboring the NBD / Rho FRET pair were formed and then incubated with NDs (5 pM) prepared with only PC / PE / PS lipids using either Hexl8A or SMA200.

[0031] FIGs. 16A-16D depict the characterization of membrane asymmetry in DeFrNDs. FIG. 16A depicts the titration of symmetric (sym) or asymmetric (asym) DeFrNDs binding to Sec A at higher concentrations at room temperature for 10 mins, and then analyzed by clear native electrophoresis. FIG. 16B depicts the titration of symmetric (sym) or asymmetric (asym) DeFrNDs binding to SecA at lower concentrations at room temperature for 10 mins and then analyzed by clear native electrophoresis. FIG. 16C depicts a graph of asymmetric DeFrNDs (8 pM) prepared with PG / PC lipids that were kept on ice for the indicated days and then assayed for binding SecA (1 pM) by clear native electrophoresis. FIG. 16D depicts a schematic and graph of ADAM 10 activation by PS lipids. Activities of ADAM 10 were analyzed in crude membranes (native), DeFrNDs or reconstituted vesicles. Data are normalized based on results from crude membranes and shown as mean ± s.d., n = 3 independent experiments.

[0032] FIGs. 17A-17G depict the optimization and characterization of DeFrMSPs for detergent-free extraction of MalFGKi native NDs from crude membranes. FIG. 17A depicts a representative image of an electrophoresis gel of crude membranes isolated from cellsoverexpressing MalFGK? that were incubated with the indicated DeFrMSPs or DDM overnight and subjected to pull down experiments. Samples at each step were analyzed by SDS PAGE. In negative control experiments, PBS buffer with the same amount of DMF was used. Red boxes indicated the extracted MalF and MalK. Abbreviations: SM, solubilized materials; E, eluted fractions; FT, flow through fractions; P, insolubilized membrane pellets. FIG. 17B depicts a graph of evolving DeFrMSP designs based on Hex-18A for converting MalFGK proteoliposomes into NDs. Data were quantified based on the density of the MalFGKo complex band normalized to that of the Hex ISA-extracted sample. FIG. 17C depicts a graph of a SEC profile of native MalFGK2 NDs using a Superdex 200 3.2 / 300 column. FIGs. 17D-17E depict graphs of diameters of native NDs harboring MalFGF , mGluR7, and HCN1 determined from negative stain EM (FIG. 17D) and DLS (FIG. 17E). FIG. 17F depicts a pie chart of lipids associated with native MalFGK? NDs from LC-MS / MS analysis. FIG. 17G depicts a graph of ATPase activities of MalFGBG WT and mutants in native NDs. Data are shown as mean ± s.d., n = 3 independent experiments.

[0033] FIGs. 18A-18D depict the enhancement and characterization of DeFrMSPs for detergent-free extraction of mGluR7 native NDs from crude membranes. FIG. 18A depicts a graph for screening DeFrMSP designs for extracting mGluR7-eGFP from crude membranes. Solubilized samples were clarified by ultracentrifugation and extracted mGluR7 proteins were quantified by monitoring the fluorescence of eGFP. ND formation efficiencies of mGluR7 were quantified based on the GFP fluorescence emission normalized to that of DDM-solubilized samples. Data are shown as mean ± s.d., n = 3 independent experiments. FIG. 18B depicts a representative image of an SDS-PAGE gel of extracted mGluR7 NDs with different DeFrMSP designs that were subjected to pull down experiments. Samples at each step were analyzed by SDS-PAGE and in-gel fluorescence imaging. Abbreviations: 1, Hex-18A; 2, Hex20B; 3, Hex20BlWA; 4, Hex20BF4W. FIG. 18C depicts a graph of the fractionation of mGluR7 native NDs using a Superdex 200 3.2 / 300 column. FIG. 18D depicts a representative negative stain EM micrograph of mGluR7 native NDs formed by Hex20BlWA. Scale bar, 30 nm.

[0034] FIGs. 19A-19F depict the optimization and characterization of DeFrMSPs for detergent- free extraction of HCN1 native NDs from crude membranes. FIG. 19A depicts a graph of the screening DeFrMSP designs for extracting HCNl-eGFP from crude membranes. Solubilized samples were clarified by ultracentrifugation and ND formation efficiencies ofHCN1 were quantified based on the GFP fluorescence emission normalized to that of DDM- solubilizcd samples. Inset: extracted HCN1 NDs with different DcFrMSP designs and negative control (PBS) were subjected to pull down experiments. Samples at each step were analyzed by SDS-PAGE and in-gel fluorescence imaging. Abbreviations: 1, Hex- 18 A; 2, Hex20BlWA; 3, PBS. Data are shown as mean ± s.d., n = 3 independent experiments. Abbreviations: SM, solubilized materials. FIG. 19B depicts a graph of the fractionation of HCN1 native NDs using a Superdex 200 3.2 / 300 column. FIG. 19C depicts a representative negative stain EM micrograph of HCN1 native NDs formed by Hexl8A. Scale bar, 30 nm. FIG. 19D depicts a graph of the extraction of HCN 1 native NDs using the indicated polymers. The majority of the extracted proteins could not be purified through affinity purification. Data are shown as mean ± s.d., n = 3 independent experiments. FIG. 19E depicts a representative image of TLC analysis of lipids in the indicated native NDs from extracted samples (left) or after purification (right). FIG. 19F depicts a graph of the quantification of PE / PC lipid ratios for the indicated extracted and purified samples. Data are shown as mean ± s.d., n = 3 independent experiments.

[0035] FIG. 20 depicts a schematic of the extracted lipidome from HEK293 cells by DeFrNDs. Crude membranes of HEK293 cells with or without treatment of DeFrMSPs (Hexl8A and Hex20BlWA) were characterized by lipidomic analysis. All lipid species identified in the crude membranes were also found in DeFrNDs. n = 2 independent experiments.

[0036] FIG. 21 depicts a schematic and representative images of the extraction of membrane proteins located in intracellular membranes by DeFrNDs. Purified secretory vesicles and ER microsomes were treated with PBS (negative control), DDM (positive control), and DeFrMSPs (Hexl8A or Hex20BlWA). Samples were then clarified by ultracentrifugation and soluble supernatants were analyzed by SDS-PAGE and western blot using antibodies against either VAMP2 or Sec6ip.

[0037] FIG. 22 depicts a schematic of one-step reconstitution of native NDs using DeFrMSPs. Traditional ND reconstitution requires the purification of the membrane protein of interest in detergent micelles (dash line) and optimization of experimental procedures to assemble with MSPs and lipids upon the removal of detergents. In contrast, the materials and methods described herein can directly extract membrane proteins into NDs with native lipids in one step, bypassing the need and limitation of detergent-mediated reconstitution.DETAILED DESCRIPTION

[0038] The details of various aspects of the invention arc set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.

[0039] Nanodiscs have a variety of applications, including the isolation of membrane proteins from cell membranes. The hydrophobic face of membrane scaffold proteins (MSPs) encircles a nanoscale patch of lipid bilayers inside nanodiscs for the reconstitution of membrane proteins, whereas the hydrophilic face ensures solubility of the reconstituted particles. A difficulty in obtaining nanodiscs with intact membrane proteins stems from reliance on the self-assembly of lipids, MSPs, and membrane proteins of interest upon the slow removal of detergents17. A prerequisite of this assembly process is to obtain stable membrane proteins in detergent micelles. Unfortunately, some membrane protein complexes often fall apart or aggregate once extracted from lipid bilayers using detergents. Chemical crosslinkers may be employed to increase the stability and homogeneity of membrane protein complexes for structural studies18, 19. However, these crosslinked protein complexes are not suitable for other biochemical and functional interrogations because their dynamics are markedly distorted.

[0040] The present disclosure provides novel peptides (e.g., isolated peptides, referenced herein as “DeFrMSPs”) that are capable of producing nanodiscs without the need for detergent. Nanodiscs produced using the DeFrMSPs described herein, in various aspects, maintain integral membrane proteins in functional states and provide an appropriate lipidic environment for the characterization of peripheral membrane proteins. Additionally, the approach described herein allows for, e.g., the preservation of a wide variety of membrane protein complexes (such as, but not limited to, transporters, receptors, and ion channels), many of which are disrupted by detergents. Also provided are methods for generating the peptides of the disclosure, methods of obtaining nanodiscs, as well as methods for isolating cellular proteins, such as membrane proteins (e.g., detergent-sensitive membrane proteins), utilizing the peptides of the present disclosure. In various embodiments, the methods of the present disclosure do not comprise a reconstitution step using detergent.

[0041] In aspects, the disclosure provides a peptide comprising an amino acid sequence of formula I:DWX1KAFYDKX2AEKX3KEAX4 (I) (SEQ ID NO: 41 ) wherein:Xi is an amino acid with hydrophobic side chain;X2 is selected from V or W;X3 is selected from L or W;X4 is selected from F or W; and wherein the N-terminus of the peptide is modified with a fatty acid, such as hexanoic acid.

[0042] Optionally, the amino acid with hydrophobic side chain (Xi) is selected from A, V, I, L, M, F, Y, or W. In various aspects, Xi is A. In various aspects, Xi is V. In various aspects, Xi is I. In various aspects, Xi is L. In various aspects, Xi is M. In various aspects, Xi is F. In various aspects, Xi is Y. In various aspects, Xi is W. In various aspects, X2 is V. In various aspects, X2 is W. In various aspects, X3 is L. In various aspects, X3 is W. In various aspects, X4 is F. In various aspects, X4 is W.

[0043] Optionally, the peptide of the disclosure comprises an amino acid sequence of formula II:DWX1KAFYDKX2AEKX3KEAX4X5W (II) (SEQ ID NO: 42) wherein X5 is selected from D or E, and X1-X4 are described above. In various aspects, X5 is D. In various aspects, X5 is E. The N-terminus of the peptide comprising amino acid sequence of formula II is modified with a fatty acid, and optionally comprises a hexanoic acid.

[0044] In various aspects, the peptide of the disclosure does not comprise SEQ ID NO: 1. SEQ ID NO: 1 (also referred to herein as “18A”) comprises the sequence DWLKAFYDKVAEKLKEAF, which has neither an N- nor a C-terminal chemical modification.

[0045] In various aspects of the disclosure, the peptide comprises the sequence DWLKAFYDKVAEKWKEAFDW comprising a hexanoic acid modification at its N-terminus (SEQ ID NO: 34).

[0046] In various aspects, the peptide comprises an amino acid sequence of formula III:DWX1KAFYDKX2AEKX3KEAX4 X5W X6K (TIT) (SEQ TD NO: 43) wherein X<> is selected from L or F, and X1-X5 are described above. In various aspects, Xe is L. In various aspects, Xe is F. The N terminus of the peptide is modified with a fatty acid, such as hexanoic acid.

[0047] In various aspects, the peptide of the present disclosure comprises a chemical modification at the N- and / or C- terminus. For example, in various aspects of the disclosure, the peptide is amidated at the C-terminus.

[0048] Further, the disclosure provides a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 22-40. Exemplary peptides of the disclosure are provided in Table 1 .Table 1

[0049] The disclosure contemplates a peptide comprising the amino acid sequence of any one of SEQ ID NO: 7-40, including a peptide comprising the amino acid sequence of any one of SEQ ID NO: 22-40, as well as peptides comprising one, two, three, or four amino acid substitutions within the amino acid sequences set forth herein.

[0050] The disclosure further provides a method of preparing a nanodisc, the method comprising (a) contacting a lipid bilayer comprising a payload with a peptide comprising an amino acid sequence of formula I; and (b) purifying a nanodisc comprising the payload. The method does not comprise a reconstitution step using detergent.

[0051] The disclosure further provides a method of preparing a nanodisc, the method comprising (a) contacting a lipid bilaycr that docs not comprise a payload with a peptide comprising an amino acid sequence of formula I; and (b) purifying a nanodisc that does not comprise the payload. The method does not comprise a reconstitution step using detergent.

[0052] Cells expressing the payload in membranes will typically be homogenized and subjected to ultracentrifugation to isolate cell membranes. These membrane fractions will be resuspended in PBS buffer and incubated with the design DeFrMSPs on ice for 20 hours with gentle shaking. The resulting samples are then optionally clarified by ultracentrifugation.Nanodiscs harboring the payload are isolated by any suitable method, including, e.g., performing affinity purification using the supernatant from ultracentrifugation and further purifying through size-exclusion chromatography to remove aggregated materials.

[0053] The amino acid sequence of formula I is DWX1KAFYDKX2AEKX3KEAX4 (SEQ ID NO: 41), wherein Xi is an amino acid with hydrophobic side chain; X2 is selected from V or W; X3 is selected from L or W; and X4 is selected from F or W. The N-terminus of the peptide is modified with a fatty acid, such as hexanoic acid. Optionally, the peptide of the method comprises an amino acid sequence of formula 11 (DWX1KAFYDKX2AEKX3KEAX4X5W (11) (SEQ ID NO: 42)), wherein X5 is selected from D or E, and the N-terminus is modified with a fatty acid. In various aspects, the peptide of the method does not comprise SEQ ID NO: 1.

[0054] The N-terminus of the peptide described herein, including the peptide used in the method disclosed herein, comprises a fatty acid modification at the N-terminus. Fatty acids are carboxylic acids comprising long aliphatic chains. In addition to conjugation to the N-terminus, fatty acids may be conjugated to the sidechain of lysine or via cysteines in the protein sequence.

[0055] The phrase “fatty acid modification” or “modified by a fatty acid” refers to conjugation (or attachment) of a fatty acid moiety to the peptide. Various approaches are available for fatty acid conjugation. The fatty acids suitable for fatty acid conjugation include, but are not limited to, caprylic acid (C8, also called octanoic acid), capric acid (CIO, also called decanoic acid), lauric acid (C12, also called dodecanoic acid), myristic acid (C14, also called tetradecanoic acid), palmitic acid (C16, also called hexadecanoic acid), and stearic acid (C18, also called octadecanoic acid). Examples of fatty acids suitable for use in the context of the disclosure include, but are not limited to, a myristoyl moiety, decanoic acid, a palmitoyl moiety, orhexanoic acid. Tn various aspects, the fatty acid modification of the peptide is modification with hexanoic acid. Hexanoic acid, also known as caproic acid, is the carboxylic acid derived from hexane with the chemical formula CH3(CH2)4COOH. Hexanoic acid is a C6, straight-chain saturated fatty acid. It has a role as a human metabolite and a plant metabolite. It is a straightchain saturated fatty acid and a medium-chain fatty acid. Hexanoic acid is a conjugate acid of a hexanoate.

[0056] In various aspects, the C-terminus of the peptide may be modified. For example, in various aspects of the disclosure, the peptide is amidated at the C-terminus. In some other aspects, the C-terminus of the peptide may be modified using other C-terminal modifications, e.g., without limitation, amides (NH2), N-alkyl amides, aldehydes, esters, p-Nitroanilide (pNA), and / or 7-Amino-4-Methylcoumarin (AMC), and the like.

[0057] In various aspects, the method comprises contacting the lipid bilayer with a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 22-40. Alternatively, the method comprises contacting the lipid bilayer with a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 7-21.

[0058] The method comprises contacting a lipid bilayer comprising a payload with the peptide of the disclosure. Lipid bilayers are polar membranes made of two layers of lipid molecules, often phospholipids, and are essential components of biological membranes. The lipid bilayer of the method may be an intact cellular membrane or a cell membrane fraction (i.e., disrupted fragments of cellular membranes prepared via, e.g., physical or chemical means). The lipid bilayer may also be present in an artificial cell membrane. In various aspects of the disclosure, the lipid bilayer is an asymmetric membrane. A common feature of cell membranes is a nonrandom distribution of lipid species in the lipid bilayer, which results in “lipid asymmetry.” In situations where the lipid bilayer is an asymmetric membrane or asymmetric bilayer, the nanodisc obtained from the method optionally retains membrane asymmetry. The asymmetrical distribution of lipids can have functional consequences to the cell and, as such, the retention of asymmetry is a technical advantage of the resulting nanodisc.

[0059] In various aspects, the lipid bilayer comprises a “payload” which may be any moiety of interest for extraction and incorporation into a nanodisc. The pay load may be a moiety expressed in prokaryotic cells or a moiety is expressed in eukaryotic cells. A representativepayload is a protein, such as a membrane protein. Examples of a payload include, but are not limited to, a transporter protein, a receptor, or an ion channel. In various aspects, the payload is a transporter protein. In various aspects, the payload is a receptor. In various aspects, the payload is an ion channel. Optionally, the payload is a detergent sensitive protein. The method of the disclosure offers a technical advantage of incorporating proteins into nanodiscs without the need for detergent in a reconstitution step, thereby allowing generation of nanodiscs with proteins which may otherwise undergo conformation change or degradation in methods wherein detergent is required for nanodisc formation, as described further below.

[0060] The nanodisc of the present disclosure may be any suitable size, optionally from about 5 nm to about 100 nm, from about 6 nm to about 60 nm, from about 8 nm to about 40 nm, from about 10 nm to about 35 nm, or from about 11 nm to about 30 nm in diameter. In various aspects, the nanodisc of the present disclosure is from about 5 nm to about 50 nm in diameter. For instance, the nanodisc may be about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, or about 100 nm in diameter.

[0061] The method of preparing nanodiscs described herein does not comprise a reconstitution step using detergent. Detergents are a class of molecules whose unique properties enable manipulation (disruption or formation) of hydrophobic-hydrophilic interactions among molecules in biological samples. Detergents are amphipathic molecules, meaning they contain both a non-polar "tail" having an aliphatic or aromatic character and a polar "head". Ionic character of the polar head group forms the basis for the broad classification of detergents; they may be ionic (charged, either anionic or cationic), non-ionic (uncharged), or zwitterionic (having both positively and negatively charged groups but with a net charge of zero). Some common detergents used in protein extraction include Sodium dodecyl sulphate (SDS), Triton X-100, Triton X-l 14, NP-40, polysorbates (such as Tween® 20 and Tween® 80), CHAPS (3-((3- cholamidopropyl) dimethylammonio)-! -propanesulfonate), and CHAPSO (3-([3- Cholamidopropyl]dimethylammonio)-2-hydroxy-l-propanesulfonate).

[0062] Detergent is often used in methods of preparing nanodiscs; yet the presence of detergent is associated with significant challenges in isolating intact proteins which mimic theirnative structure and function. Membrane proteins are often modified and inactivated by detergent solubilization as a result of native lipid interactions having been disrupted. Removing detergents from the milieu is often complicated, if not impossible, depending on the structure and complexity of the specific membrane protein to be extracted. The method described herein advantageously bypasses the use of detergent in a reconstitution step, thus avoiding the complications associated with detergent use in this context.EXAMPLES

[0063] The examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0064] Representative materials and methods for generating the nanodisc of the disclosure, as well as further characterizing various features of the nanodisc of the disclosure, are described below.Materials and MethodsChemicals and Reagents

[0065] l,2-dioleoyl-sn-glycero-3-phosphocholine (PC), l,2-dioleoyl-sn-glycero-3-phospho-L serine (PS), l,2-dioleoyl-sn-glycero-3-phospho-(l'-rac -glycerol) (PG), 1,2-dioleoyl-sn-glycero- 3-phosphoethanolamine-N-(7-nitro-2-l ,3-benzoxadiazol-4-yl (NBD-PE), and 1 ,2-dioleoyl-sn- glyccro-3-phosphocthanolaminc-N-(lissaminc rhodamine B sulfonyl (Rho-PE) were obtained from Avanti Polar Lipids. Nitrilotriacetic acid (Ni2+NTA)-chelating Sepharose and Superose 6 increase 10 / 300 GL were purchased from GE Healthcare. l,l'-Dioctadecyl-3,3,3',3'- Tetramethylindocarbocyanine Perchlorate) (Dil), N,N'-Dimethyl-N-(Iodoacetyl)-N'-(7- Nitrobenz-2-Oxa-l,3-Diazol-4-yl)Ethylenediamine (IANBD amide) and Oregon Green™ 488 (OG) maleimide were obtained from ThermoFisher. All other chemicals were acquired from Sigma.Plasmids

[0066] pTrc-MalFGK2 is described in Bao, H. & Duong, F. Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation. The Journal of Biological Chemistry 289, 9844-9851 (2014). pEThis-vamp2 is described in Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759-772 (1998). pGEX-sytland pDuetRSF t-dimer are described in Bao, H. et al. Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties. Nature 554, 260-263 (2018); and Bai, H. et al. Different states of synaptotagmin regulate evoked versus spontaneous release. Nature Communications 7 (2016). All other constructs described in the instant disclosure were made using the In-Fusion® HD Cloning Kit (Takara Bio USA).Proteins and Peptides

[0067] Protein expression and purification for sytl, MBP, MalFGKi, and soluble N- ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins were performed as described previously33, 41, 42, 58"60. Briefly, plasmids were transformed into BL21 cells that were grown in LB supplemented with Km (50 mg / ml) or Amp (100 mg / ml) to ODeoo ~0.7. Protein expression was induced with 0.2 mM isopropyl P-D-l -thiogalactopyranoside (IPTG) at 16 °C overnight. Bacteria were harvested by centrifugation at 3,700 rpm for 20 mins, resuspended in Buffer A (50 mM Tris-HCl (pH 8), 100 mM NaCl, 5% glycerol, 2 mM P-mercaptoethanol), and lysed using a Branson cell disrupter. Cell lysates were clarified by centrifugation at 10,000 rpm for 45 mins. For sytl and MBP, the supernatants were loaded onto a 1 ml nitriloacetic acid (NTA) column (GE Healthcare), followed by two times wash using buffer B (50 mM Tris-HCl (pH 8), 20 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM P-mercaptoethanol). Proteins were eluted in buffer C (50 mM Tris-HCl (pH 8), 500 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM P-mercaptoethanol), desalted in buffer A using PD MiDiTrap G-25 (GE Healthcare), and stored at -80 °C.

[0068] Ecto-ELFNl-Fc (residues 1-399) was expressed as previously described62. An eGFP fusion was added to the C-terminus of mGluR7 and expressed62. Briefly, mGluR7 and Ecto- ELFNl-Fc constructs were inserted into modified pEG BacMam vector74. The bacmid was then transfected into Sf9 cells (ThermoFisher Scientific, 11496015) to produce baculovirus. The supernatant of the third generation of virus production (P3) was then harvested and added to HEK293S GnTi- cells (ATCC, CRL-3022) grown at 37 °C with 8% CO2 at a confluency of 2.0- 3.0xl06cells / mL in FreeStyle 293 expression media. After 18 hours, sodium butyrate (Sigma- Aldrich, B5887) was added to 10 mM. The cells were then incubated for a further 48 hours and pelleted by centrifugation. For the purification of ELFN-1, the supernatant of cell lysates clarified by centrifugation (8, 184 x g, 1 h) was then affinity purified using Pierce™ anti-DYKDDDDK affinity resin (ThermoFisher Scientific, A36801) and eluted with 3 x Flag peptide (GcnScript, RP21087), concentrated using an Amicon™ Ultra-4 centrifugal filter (Sigma- Aldrich, UFC805024), analyzed by SDS-PAGE, aliquoted, and flash frozen.

[0069] For MalFGK2 and SNAREs, membrane fractions were resuspended in Buffer A and solubilized with 1% DDM (4 °C, overnight). Solubilized membrane extracts were isolated by centrifugation at 50,000 rpm for 45 mins. Supernatants were loaded onto a 1 ml NTA column (GE Healthcare), followed by two times wash using buffer B (50 mM Tris-HCl (pH 8), 20 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM P-mercaptoethanol, 0.02% DDM). Proteins were eluted in buffer C (50 mM Tris-HCl (pH 8), 500 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM P-mercaptoethanol, 0.02% DDM), desalted in buffer A supplemented with 0.02% DDM using PD MiDiTrap G-25 (GE Healthcare).

[0070] To express mGluR7, the HEK293 GnTI- cells were grown at a density of 3.0 x 106per mL and infected with high titer P3 BacMam viruses. 10 mM sodium butyrate was added to the culture to enhance the expression of proteins after 12 h post-infection. The culture was further incubated at 30 °C for 48 hr and harvested by centrifugation.

[0071] To express HCN1, cDNA corresponding to HCN1SM in the modified pEG BacMam vector was extracted from large volumes of bacterial cultures using Endotoxin free Plasmid Purification kits (Qiagen) and transfected into suspension cultures of Freestyle HEK 293 cells (Thermo Fisher Scientific) using Trans-IT Pro Transfection reagent (MIRUS) following manufacturer’s instructions. The Freestyle HEK93 cells were not experimentally authenticated or tested for mycoplasma contamination. Post transfection, cells were grown at 12-14 hr at 37 °C, following which sodium butyrate was added to the cultures to a final concentration of 10 mM and cultures were grown at 30 °C for another 48 hr. Cells were collected by centrifugation at 3,000g for 20 min and washed twice with chilled 150 mM NaCl, 2 OmM Tris, pH 8.0. Cell pellets were subsequently resuspended in lysis buffer (300 mM NaCl, 40 mM Tris, 10 mM DTT, 20% glycerol, 1 mM EDTA, 2 mM cholesterol hemi succinate (CHS), pH 8.0 supplemented with IX Halt protease inhibitor cocktail (Thermo Fisher Scientific)) and stored at -80°C.

[0072] All peptides were custom synthesized by GenScript with 95% purity. Peptides were stored at -20 °C and dissolved at 100 mg / ml in DMF before each use.Purification of dense-core vesicles

[0073] PC 12 cells were cultured in Dulbecco’s modified Eagle’s medium, high glucose(Gibco) supplemented with 10% horse scrum (CcllGro), and 10% calf scrum (Fc+) (Hyclonc), with 1% penicillin / streptomycin mix. Cell media was changed every 2 to 3 days and split when cells reached -90% confluency by incubating for 5 min in Hank’s balanced salt solution, resuspended by adding fresh medium, and replating them at a 6:1 ratio. Dense core vesicles were then purified from 15 10-cm plates (90% confluent) by scraping them into phosphate buffered saline (PBS) solution. Cells were pelleted by centrifugation at 1000 x g for 5 min, resuspended, and washed once by repeating the centrifugation step in homogenization media (0.26 M sucrose, 5 mM MOPS, and 0.2 mM EDTA). Cell pellet was resuspended in 3 mL of homogenization medium containing protease inhibitor (Roche Diagnostics), the cells were then physically lysed by passing cells through a ball bearing homogenizer 10 times, which contained a 0.6367 cm bore and a 0.6340 cm diameter ball. Nuclei and large debris were removed by centrifugation in a fixed angle microcentrifuge at 1000 x g for 10 min at 4 °C. The post-nuclear supernatant was collected, and mitochondria were removed by centrifugation at 8,000 x g for 15 min at 4 °C. The post mitochondria supernatant was then collected and adjusted to 5 mM EDTA and incubated on ice for 10 min to assist with removing ribosomes from the endoplasmic reticulum to reduce contamination in the dense core vesicle fraction. A working solution of 50% OptiPrep (iodixanol) was made by mixing five volumes of 60% OptiPrep with one volume 0.26 M sucrose, 30 mM MOPS, and 1 mM EDTA. This working solution was mixed with homogenization media to prepare solutions of 30% iodixanol and 14.5% iodixanol for a discontinuous gradient. A SW55 tube was prepared by laying 3.8 mL of 14.5% iodixanol on top of 0.5 mL of 30% iodixanol. Then 1.2 mL of the post mitochondrial supernatant was layered on top of this iodixanol gradient. The sample was then centrifuged at 190,000 x g for 5 hours. A clear white band at the interface between the 30% iodianol and 14.5% iodixanol was collected as the dense core vesicle sample. The dense core vesicles sample was then extensively dialyzed in a cassette with 10,000-kDa molecular weight cutoff (24 h with 3 buffer changes of 5 L each) into 120 mM potassium glutamate, 20 mM potassium acetate, and 20 mM HEPES, pH 7.4. After dialysis, sucrose was spiked in to make a final concentration of 10% sucrose and then the sample was flash frozen and stored at -80 C.Fluorescent Labeling of Proteins

[0074] Purified sytl was desalted using Zeba Spin columns (Thermo Fisher) in buffer D (50 mM Tris-HCl, pH 8, 100 mM NaCl, 5% glycerol) and labeled with a 3-fold excess of IANBD amide or OG maleimide in the presence of TCEP (0.2 mM) at room temperature for 2 hours. Free dyes were removed by passing through Zeba Spin columns in buffer A.Preparation for proteoliposomes and liposomes

[0075] Lipids were dried under a gentle stream of nitrogen and further with vacuum for 2 h followed by rehydration in reconstitution buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, ImM DTT). Samples were then extruded through a 200 nm filter for liposome preparation. To monitor the binding of sytl to protein-free NDs via fluorescence spectrometry, protein-free liposomes were made using 70% PC and 30% PS. To study the interaction of SecA with lipids, protein-free liposomes were made using 100% PC, 100% PG, or 50% PC with 50% PG. The formation of asymmetric liposomes with PC / PG lipids was induced by generating a transmembrane pH gradient as described previously57. For pro teolipo some reconstitution, purified proteins (MalFGBG and SNAREs) and lipids were incubated at a ratio of 1:1000 in reconstitution buffer plus 1% OG on ice for 30 mins. MalFGK proteoliposomes were prepared with 100% PC lipids. For membrane fusion and lipid exchange assays, donor v-SNARE and protein-free vesicles were prepared with 12% PE, 40% PS, 45% PC, 1.5% NBD-PE and 1.5% Rho-PE, whereas t-SNARE and protein free vesicles were prepared with 15% PE, 25% PS and 60% PC. Detergents were removed by addition of Bio-beads (1 / 3 volume) and gentle shaking (O / N). Finally, proteoliposomes were purified by flotation in an Accudenze step gradient as described previously49.Nanodiscs

[0076] The peptides of the present disclosure are referenced herein as “DeFrMSPs.” To generate nanodiscs, candidate DeFrMSPs (candidate peptides) were incubated with membranes at 4 °C overnight with gentle shaking. The resulting materials were clarified by ultracentrifugation. Samples were then loaded onto affinity and size exclusion column to isolate the nanodiscs.

[0077] Candidate DeFrMSPs (100 mg / ml in DMF) were first diluted to 25 mg / ml in PBS and added to proteoliposomes at 1 mg / ml or crude membranes at 4 mg / ml. Samples were incubated at 4 °C overnight and clarified by centrifugation at 50,000 rpm for 45 mins. Nanodiscs werepurified by affinity purification as described above, and further fractionated by size-exclusion chromatography (SEC) and stored at -80 °C. To monitor the binding of sytl to lipids via fluorescence spectrometry, lipids were made using 70% PC and 30% PS. For the reconstitution of membrane fusion, lipids were prepared using 1% NBD-PE, 1% Rho-PE, 20% PE, 48% PC, and 30% PS. To study the interaction of SecA protein with lipids, nanodiscs were made using 50% PC and 50% PG.

[0078] To directly reconstitute membrane proteins into DeFrMSP native nanodiscs, cells were lysed using a cell disrupter (Fisher) and then isolated crude membranes were obtained by ultracentrifugation (184,000 x g, 1 h). These membrane preparations (~10 mg / ml) were resuspended in buffer A and then incubated with DeFrMSP at 1 mg / ml (4 °C, O / N). Extracted samples were clarified by ultracentrifugation (112, 000 x g, 1 h) and supernatants were subjected to affinity purification. MalFGFG NDs were purified using Ni2+-NTA beads as described above. mGluR7 NDs were purified using Ni2+-NTA and anti-DYKDDDDK (Fisher) affinity resins as ELFN-1. HCN1 NDs were purified using Streptactin affinity resin (IBA Life Sciences) and eluted in buffer D (50 mM Tris-HCl (pH 8), 300 mM NaCl, 10% glycerol, 2 mM DTT) plus 5 mM desthiobiotin. Finally, MalFGKo and mGluR7 NDs were further fractionated on a Superdex S200 3.2 / 300 column in buffer A, whereas HCN1 NDs were in buffer D. The extraction of Sec61B into DeFrNDs was assayed from Canine pancreatic ER microsomes. ER microsomes were incubated with DeFrMSPs as for crude membranes described above. ER solubilized microsomes were then spun down for 10 minutes at 12,000 x g at 4°C in a Sorvall Legend X1R tabletop centrifuge. The pellet was discarded, and the supernatant was subjected to western blot analysis using a Sec61B antibody (Novus Biologicals, NBP2-13290) and a goat anti-rabbit secondary antibody (ThermoFisher, A32735). To evaluate the extraction of VAMP2 into DeFrNDs from dense-core vesicles, samples were incubated with DeFrMSPs. Extracted samples were clarified by centrifugation at 112,000 x g at 4°C for Ih and then analyzed by western blot using a VAMP2 antibody (Synaptic systems, 104211) and a secondary mouse IgG Fc binding protein (Santa Cruz Biotechnology, sc545209). To generate polymer NDs, MalFGFG proteoliposomes, protein-free liposomes, or HCN1 crude membranes were incubated (4 °C, O / N) with the indicated polymers at a final concentration of 1 % as recommended by the vendor (Curi Bio). Samples were clarified by ultracentrifugation (112, 000 x g, 1 h). For proteoliposomes and protein-free liposomes, extracted NDs were then purified by SEC using Superdex 20010 / 300 in buffer A. For extracted HCN1 NDs from crude membranes, samples were subjected to affinity purification using Strcptactin affinity resin as described above for DcFrNDs.Negative Stain Electron Microscopy

[0079] Formvar / carbon-coated copper grids (01754-F, Ted Pella, Inc.) were glow discharged (15 mA, 25 secs) using PELCO easiGlow™ (Ted Pella, Inc). Nanodiscs (NDs) (10 pg / ml) were applied onto the grids for 30 secs, followed by staining with 0.75% uranyl formate for 1 minute. Images were collected using a ThermoFisher Science Tecnai G2 TEM (100 kV) equipped with a Veleta CCD camera (Olympus). All TEM data were analyzed using Fiji to determine the nanodisc sizes.CryoEM data collection

[0080] Three pl of MalFGK2 NDs at a concentration of 2mg / ml was applied to glow discharged 300 mesh UltraAufoil gold Rl.2 / 1.3 (quantifoil) and vitrified using a Vitrobot Mark IV (Thermo Fisher Scientific / FEI) at 10 °C and 100% humidity with a blot time of 4s, and a blot force of 0. Cryo-EM data for single particle analysis were collected at New York Structural Biology Center on a 300kV Titan krios electron microscope (Thermo Fisher Scientific / FEI) with a Gatan K3-Bioquantum direct electron detector (Gatan, Inc.) and energy filter, using leginon software75. Movies were captured at a magnification of 81,000x (pixel size of 1.058 A). A total of 12522 movies in the defocus range of -0.8 to -2.2 pM were recorded with a total accumulated dose of 55.80 e- / A.Mass Spectrometry

[0081] Lipids were extracted from nanodiscs and characterized by liquid chromatography with Waters BEH C8 column (2.1 mm x 100 mm, 1.7 um) usingl6 min gradient. Mobile phase A is 10 mM ammonium acetate with 5% methanol, 0.1% acetic acid in H2O and mobile phase B is 0.1% acetic acid in methanol. Flowrate was set at 0.4 mL / min and column oven temperature at 30 °C. 5 pL of samples were injected into Vanquish UHPLC (Thermo Scientific) connected with Orbitrap ID-X Tribrid Mass Spectrometer (Thermo Scientific). For global untargeted lipidomics, AcquireX DeepScan was applied, and data was acquired in both negative and positive ion mode. The MS resolution was set at 60,000 for both MSI and MS2 scans with scan range of 200-1100 m / z. The capillary voltage in the positive mode was 3.4 kV and the negativemode was 2.4 kV, respectively. The ion transfer tube temperature is 325 °C. The database search was performed by Compound Discoverer 3.3 with mzVault (MONA, GNPS, and NIST), mzCloud, and LipidBlast.Image processing, 3D reconstruction, modeling, and refinement

[0082] Data processing was performed using cryoSPARC v4.4.176. Raw movies were aligned using patch motion correction, and the micrograph contrast transfer function (CTF) parameters were estimated via patch CTF estimation. Micrographs were picked using a blob picker, and an initial particle set was chosen through 2D classification. Selected 2D classes were used for templet particle picking. Ab-initio jobs were run with 4 classes, and one of the best classes was used for further data processing. Three rounds of heterogeneous refinement were employed to remove junk particles. Details of the Cryo-EM classification and processing are shown in FIGS. 14A-14D. The resulting curated particle sets were used for local CTF refined to high A resolution using non-uniform refinement with 3.26 resolution using the 0.143 Fourier Shell Correlation criterion.

[0083] A model was built starting from PDB structure of truncated 7t4e. The initio model was rigid body docked into the density and adjusted in Chimera77 and coot78. Loops were manually fit into the density map using coot. Subsequently, the real space refinement was performed, with remaining manual adjustments performed in Coot. Models were validated using Molprobity in Phenix79. A summary of model refinement statistics is provided in Supplementary Table 2.Supplementary Table 2: Cryo-EM data collection, refinement, and validation statisticsLipidomics

[0084] NDs or crude membranes were subjected to lipid extraction by MTBE (methyl tert- butylether) by tip sonication three times and vortex mixing three times, and the upper phase containing the lipids was collected and dried by SpeedVac with no heat. Dried samples were reconstituted with 50 uL of 50% EtOH and used for the LC-MS / MS using Vanquish Horizon UHPLC (Thermo Scientific) and Orbitrap Tribrid ID-X (Thermo Scientific) mass spectrometry. Database search was carried out by Compound Discoverer 3.3 SP2.Fusion Assays

[0085] t-SNARE vesicles encapsulated with glutamate and fluorescence dextran were prepared as described previously41, 42. Fusion assays were performed by incubation of v-SNARE nanodiscs (0.5 pM) and t-SNARE vesicles (1 pM) in reconstitution buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl) at 37 °C for 30 mins. For the glutamate release assay, the glutamate sensor iGluSnlR61(0.1 pM) was added to the reaction mixture. For the dextran release assay, samples were centrifuged at 50,000 g for 30 min, and the supernatants were carefully collected for further analysis. The fluorescence of iGluSnFR and dextran was quantified using a Synergy HIM plate reader with excitation at 460 nm and emission at 530 nm. The percentages of cargo release weredetermined by normalization of data to the maximal release after addition of 0.5% DDM to each sample.Fluorescence Spectroscopy

[0086] NBD-labeled sytl (10 nM) was incubated with nanodiscs at the indicated concentrations. The fluorescence spectrum of samples was collected on a Synergy HIM plate reader with excitation at 460 nm and emission from 500 to 650 nm.Electrophoresis / chromatography

[0087] SDS-PAGE and Native PAGE electrophoresis were performed using 12% or 4-15% TGX stain-free™ protein gels (Bio-Rad). Size-exclusion chromatography (SEC) was carried out using Superdex 200™ increase 3.2 / 300 or 10 / 300 (GE Healthcare) in 50 mM Tris-HCl, pH8, 100 mM NaCl, 5% Glycerol33'59. TLC was performed as described previously in Bao et al.Discovery of an auto-regulation mechanism for the maltose ABC transporter MalFGK2. PloS One 1, e34836 (2012).Single molecule cAMP binding measurements

[0088] Single molecule cAMP binding measurements were done using zero-mode waveguides fabricated as described previously63. Imaging was done on a custom micromirror TIRF setup (Madcity labs) equipped with a NA 60X oil immersion objective lens (Olympus). Micromirror TIRF excitation field generated cither with a 488nm or 561nm lasers (OBIS, coherent) to excite the GFP and fcAMP, respectively. The subsequent fluorescence emission was recorded on a 512x512 EMCCD camera (Andor iXON Ultra) at a frame rate of 10Hz. Data collection was done using Micromanager 2.0. The emission was passed filtered through a dichroic filter (T5651pxr Chroma) and then a band-pass filter (Chroma ET550 / 25 nm) for GFP and a band-pass filter (Semrock bright line 593 / 40 nm) for fcAMP were in the corresponding emission pathways. The binding data were recorded from each molecule for a minimum of 240 seconds with 100 msec exposure time.

[0089] Single-molecule data was analyzed as described63. Briefly, single molecule traces were extracted and analyzed using the DISC software for the colocalization and image projection to obtain time-dependent fluorescence intensity changes. The traces were then idealized using the DISC software. The equilibrium constant for intrinsic binding affinity, K, was calculatedfrom the dwell times corresponding to the singly-bound state as described previously using QUB software63. Assuming that the binding of each of the four ligands binds to the channel independently, the binding curve was generated by calculating microscopic rate constants kl=K / 4, k2=2 / 3(K), k3=3 / 2(K), and k4= 4K and substituting them in Adair’s equation81.ADAM 10 reconstitution and biochemical assays

[0090] Crude cell membranes were prepared from HEK293S GnTi- cells (ATCC, CRL-3022) grown to a confluency of 2.0-3.0xl06cells / mL and converted to DeFrNDs using Hexl8A (1 mg / ml, 4°C, O / N) as described above for mGluR7. Samples were then clarified by ultracentrifugation (112,000 x g, Ih) and fractionated on a Superdex 200 10 / 300 column in buffer A. Soluble fractions containing ADAM 10 DeFrNDs were identified by western blot using an anti- AD AM 10 antibody (Abeam, abl997) and concentrated to ~0.5 mg / ml for biochemical assays. For detergent mediated reconstitution of ADAM10, cell membranes were solubilized with 1% OG (4 °C, O / N) and then allowed to reform vesicles by the addition of Bio-beads (1 / 3 volume) and purified by flotation in an Accudenze step gradient. Activities of ADAM 10 in DeFrNDs or vesicles formed by detergent mediated reconstitution were determined using an ADAM 10 assay kit (BPS Bioscience) in comparison with crude membranes. Data were normalized based on the amount of ADAM 10 levels measured by western blot.

[0091] The following examples describe the peptides disclosed herein and demonstrate that the peptides efficiently generate nanodiscs without the use of detergent in a reconstitution step.Example 1 - Peptides for detergent-free nanodisc reconstitution

[0092] A study was performed to characterize the ability of amphipathic peptides to extract a bacterial prototype ATP-binding cassette transporter, MalFGK , into nanodiscs from proteoliposomes (FIG. 1A). MalFGK is composed of two transmembrane proteins (MalF and MalG) and two copies of the ATPase component (MalK) that power the transport of maltose into bacteria through the interaction with the maltose binding protein (MBP)32. Previous studies have shown that the basal ATPase activity of MalFGKi in membranes is low and vastly stimulated by maltose and MBP33, 34. In contrast, the detergent-solubilized MalFGK complex is over 100-fold more active and is no longer stimulated by maltose and MBP. Thus, the ATPase activity of MalFGK? is highly sensitive to the lipid environment, serving as a useful feature to evaluate the performance of membrane mimetic reconstitution systems.

[0093] A panel of amphipathic peptides ranging from ApoA-mimicking peptides to antimicrobial peptides was used for this experiment (FIG. 7A and Table 2).Table 2[00941 Formation of MalFGKF nanodiscs from direct incubation of proteoliposomes with peptides was characterized using blue-native electrophoresis (native-PAGE) (FIG. IB and FIG. 7A). Once extracted from lipid bilayers into micelles or incorporated into nanodiscs, MalFGK would migrate as a single membrane protein complex with a molecular weight of approximately 200 kDa, as shown with the detergent DDM. This simple readout allowed for rapid characterization of more than a dozen amphipathic peptides for detergent-free reconstitution of MalFGK2 into nanodiscs. The results showed that several ApoA-mimicking peptides (e.g., 18A27, 22A12, 4F25, and NSP26) were able to transform MalFGK proteoliposomes into nanodiscs as compared to other amphipathic or antibacterial peptides. However, these nanodiscs containeda mixture of monomer, dimer, and different oligomers of MalFGK since multiple bands of protein complexes with higher molecular weights were observed on the gel.

[0095] Since 18A was observed to be the most effective for the extraction of MalFGKi monomer, MalFGK2 nanodiscs formed by 18A (18A-MalFGK2 nanodiscs) were purified through size-exclusion chromatography (SEC) (FIG. 1C). Soluble particles were isolated after incubating MalFGK2 proteoliposomes with 18A. In control experiments, only large, insoluble particles lacking peptides were observed. Quantitative analysis also confirmed the presence of -100 copies of lipids per ND (FIG. 12B), consistent with the idea that 18A directly transforms proteoliposomes into discoidal particles. Moreover, MalFGK2 encased in 18A NDs is functional as its ATPase activity remains coupled to maltose and MBP (FIG. ID). In contrast, MalFGBG was inactive and did not respond to maltose and MBP after extraction into NDs using amphipathic polymers (FIGs. 12C-12D). The lack of detectable ATPase activities of MalFGK in polymer NDs is probably due to the sensitivity of these polymers to bivalent cations29, causing the aggregation of the transporter in ATP hydrolysis buffer containing high concentrations of MgC12 (FIGs. 12E-12F). In addition, lipids were coeluted with these nanodiscs (FIG. 7B). Moreover, MalFGK2 was reconstituted in a functional state in these nanodiscs. The ATPase activity of MalFGK2 embedded in these nanodiscs remained coupled to maltose and MBP (FIG. ID). In contrast, MalFGK did not respond to maltose and MBP after extraction into nanodiscs using amphipathic polymers (FIG. 7C).

[0096] Next, the 18 A-MalFGK2 nanodiscs were characterized using negative stain electron microscopy (EM). Although the purified particles from size-exclusion chromatography (SEC) show diameters of about 10-20 nm (FIG. IE), they were quite poly-disperse, consistent with the data from native PAGE (FIG. IB). Further, the structural feature of the MalFGK2 transporter was difficult to identify from raw images, highlighting deficiencies with 18 A.Example 2 - Characterization of peptide fatty acid modifications in reconstituting nanodiscs

[0097] In an attempt to improve the activity of 18 A, 18A was fused with antimicrobial peptides. Peptides also were functionalized with distinct chemical groups, such as amide, acetyl, and fatty acids. To assay the efficiency of native nanodisc reconstitution, the same MaFGK2 proteoliposomes were incubated with an increasing concentration of these modified peptides and analyzed by native electrophoresis. The results showed that fatty acid modifications of 18Aprofoundly enhanced the monodispersity of MalFGF nanodiscs (FIG. 2A and FIG. 8A), migrating as a single band on non-denaturing native gels. Surprisingly, among all the fatty acid modifications, hexanoic acid functionalized 18A (Hex-18A) resulted in the highest efficiency and was selected for further characterization.

[0098] MalFGKo NDs enclosed by Hexl8A from SEC were purified and their ATPase activities were examined (FIG. 13B). Again, the transporter was fully functional and stimulated by maltose and MBP. In addition, lipids were coeluted with these NDs (FIG. 13C), as expected for the conversion of MalFGK2 proteoliposomes into -10-15 nm discoidal particles. These samples were analyzed by negative stain EM (FIGs. 13C-13D). In contrast to the results obtained with 18A, NDs formed by Hexl 8A were much more monodisperse, with clearly appreciable features of the cytosolic ATPase subunit of MalFGBG. The diameters of these MalFGK2 NDs formed with Hexl8A averaged at -12 nm, which would provide a -2.5 nm layer of PC lipids around the transporter.

[0099] N- and C-terminal modifications can enhance peptide insertion into membranes. Thus, several chemical modifications (e.g., amide, acetylene, and different kinds of fatty acids) were tested. Hexanoic acid was the shortest fatty acid modification tested, yet it was the most effective one. It was initially expected that the longer fatty acids would be more effective as they are more hydrophobic and should be more active to destabilize bilayer and form nanodiscs. However, without wishing to be bound by any particular theory, the longer fatty acids may also make the peptide too hydrophobic to stay soluble in solution. Thus, peptides containing both N- terminal Hexanoic acid modification and C-terminal amidation were less active. Thus, peptides modified only with hexanoic acid at the N-terminus described herein were shown to be the best for nanodisc formation.

[0100] MalFGK2nanodiscs enclosed by Hex-18A were purified from size-exclusion chromatography (SEC) and their ATPase activities were determined (FIG. 2B and FIG. 8B). Again, the transporter was fully functional and stimulated by maltose and MBP. Quantitative analysis also confirmed the presence of approximately 150 copies of lipids per nanodisc (FIG. 8C), as expected for approximately 10 nm discoidal particles. Negative stain EM was used to analyze these samples (FIG. 2C). In contrast to nanodiscs formed by 18A, the nanodiscs formedby Hex-18 A were much more homogeneous, with clearly appreciable features of the cytosolic ATPasc subunit of MalFGKi.

[0101] The much-improved homogeneity of the Hex-18A MalFGF nanodiscs suggested that these samples are suitable for structural studies using single particle analysis by cryogenic electron microscopy (cryo-EM). Indeed, the structure of the MalFGK2 complex in Hex-18A nanodiscs was observed (FIG. 2D and FIG. 8D), and is overall in agreement with the previous crystal structure obtained using truncation in the N -termini of MalF or arrested by the glucose enzyme EIIA35, 36. Together, the results suggested that Hex-18A can extract MalFGIG into nanodiscs from membranes for structural and functional characterizations.Example 3 - DeFrMSPs enclose stable and functional membranes for biochemical reconstitutions

[0102] The stability and function of membrane mimetics vastly affect the conformational state of membrane proteins37’39. Further, the lipids in previous detergent-free nanodiscs formed by amphipathic polymers undergo rapid exchange with each other22, indicating that the integrity of the enclosed membranes is compromised and might not be able to faithfully recapture the dynamics of transmembrane signaling events. Therefore, the membrane properties in DeFrMSP nanodiscs were examined.

[0103] First, it was determined if DeFrMSP can encase reconstituted lipid bilayers into monodisperse NDs. To this end, protein-free liposomes were prepared with various synthetic lipids or membrane extracts (FIGs. 15A-15F). These samples were then incubated with increasing amounts of Hexl8A followed by fractionation on SEC (FIGs. 15A-15C). At the optimized Hexl8A / lipid ratio, well-defined ~15 nm NDs were formed with all the lipid mixtures tested in these experiments. Interestingly, the formation of 30-50 nm NDs was observed at lower Hexl8A / lipid ratios, indicating that the diameter of DeFrMSP NDs is flexible and potentially programmable.

[0104] A classic FRET assay was employed to determine if the lipids in DeFrMSP nanodiscs are stably embedded or rapidly diffused among individual nanodiscs40. In these experiments (FIG. 3A), donor nanodiscs were prepared with a FRET pair (NBD-PE and Rho-PE) and then incubated with acceptor DeFrMSP nanodiscs or liposomes that do not harbor fluorescent lipids41,42. If the lipids in DeFrMSP nanodiscs are unstable, the FRET pair, upon incubation withacceptor nanodiscs or liposomes, will be diluted and separated from each other, resulting in the increase of the fluorescence of NBD-PE. However, only a negligible increase in NBD fluorescence was observed after incubating donor DeFrMSP nanodiscs with acceptor nanodiscs or liposomes (FIG. 3B), similar to the negative control conditions using protein-free donor liposomes. In positive control experiments, the classic membrane fusion machinery, soluble N- ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs), was employed. Specifically, the cognate vesicle (v) and targeted membrane (t) SNAREs were reconstituted into donor nanodiscs and acceptor liposomes, respectively. Fusion between v-SNARE nanodiscs and t-SNARE liposomes readily caused the dequenching of NBD fluorescence. Thus, the membranes in DeFrMSP nanodiscs were stably enclosed and were also suitable for the biochemical reconstitution of membrane fusion.

[0105] Additionally, a study was conducted to assess if the DeFrMSP nanodiscs can functionally capture protein-lipid interactions. For this purpose, nanodiscs were prepared with different lipid mixtures for the characterization of membrane binding and remodeling by synaptotagmin- 1 (sytl), the Ca2+sensor responsible for synaptic transmission43-45. Using a well- established fluorescent-based assay46, sytl-lipid interaction will result in fluorescence increase in a Ca2+and negatively charged lipids-dependent manner; this was observed using DeFrMSP nanodiscs (FIG. 3C). The results establish that the membranes in DeFrMSP nanodiscs are stable and functional for biochemical reconstitution of membrane biology.Example 4 - Reconstitution of asymmetric membranes in DeFrMSP nanodiscs

[0106] One distinct feature of native membranes is lipid asymmetry that is critical for numerous cell signaling pathways47, 48. However, the recapturing of this lipid asymmetry in nanodiscs is challenging because detergent-mediated reconstitution will disrupt the structure of membranes. Nanodiscs prepared using the DeFrMSP described herein were assayed in the context of asymmetric membranes.

[0107] Asymmetric PC / PG liposomes were formed using a previously described pH-driven method49. These liposomes were incubated with DeFrMSPs to form nanodiscs. If membrane asymmetry is preserved in DeFrMSP nanodiscs, they should contain mainly PG lipids on one side and largely PC lipids on the other side. SecA protein, which binds strongly to PG lipids, was used to examine if SecA protein binds to both sides or only one side of DeFrMSP nanodiscs(FIG. 3D). SecA protein is a dimeric protein in solution and will dissociate into monomers upon binding to negatively charged lipids50. Using symmetric membranes in DcFrMSP nanodiscs with PG lipids, two bands on native gel were readily observed, corresponding to the monomer on one side, and two monomers on both sides of nanodiscs. With asymmetric membranes, the lower band of the nanodisc-SecA monomer complex is much more enriched, indicating that membrane asymmetry is reconstituted in DeFrMSP nanodiscs. The distribution of monomeric SecA binding to one or two sides of NDs were different by titrating ND concentrations (FIGs. 16A-16C). At lower ND concentrations, a significant increase of two monomeric SecA proteins was observed bound to both sides of NDs, indicating that asymmetric NDs were not 100% asymmetrical with PG lipids located on only one leaflet. This issue could arise at the stage of generating asymmetric liposomes or as a result of ND formation using DeFrMSPs.

[0108] Encouraged by the above SecA-based experiments, DeFrNDs were further examined to determine if they could maintain the structural organization of native cell membranes. For this purpose, ADAM10 was used. ADAM10 is an abundant protease expressed in many cells that is activated by PS externalization59(FIG. 16D). DeFrNDs were prepared from HEK293 cells to assay ADAM10 activities. The results showed that ADAM10 activities in DeFrNDs and crude membranes were quite similar. However, the activity of ADAM10 was significantly increased if vesicles were prepared from crude membranes using detergent-mediated reconstitution, probably because detergent completely disrupted native cell membranes and caused the redistribution of PS lipids between the two leaflets of reconstituted vesicles.

[0109] The stability of the asymmetric NDs was assessed using the SecA-ND binding assay. The results showed that the asymmetric membranes in NDs were relatively stable for a week when kept on ice (FIG. 16D).

[0110] Thus, these results establish that the detergent-free nanodiscs are suitable for the reconstitution of asymmetric membranes.Example 5 - Detergent-free reconstitution of nanodiscs from native membranes

[0111] As compared to detergent-mediated reconstitution, an advantage of the method described herein is the potential to stabilize membrane protein complexes with native lipids (FIG. 4A). To illustrate, crude membrane fractions from E. coli expressing MalFGK2 were incubated with Hex-18 A and affinity purification was performed. The results established thatM&1FGK2 was directly isolated in Hexl 8A-encased native nanodiscs (FIG. 4B). However, the results also showed that Hcx-18A was much less efficient than the detergent DDM to extract MalFGK? from crude E. coli membranes. Additional peptides were generated to achieve a DeFrMSP with superior performance to Hex-18A. The amino acid sequence of SEQ ID NO: 1 was modified with a combination of positively charged residues with hydrophobic ones. Additionally, Phe was replaced with Tip in various candidates, and impact on efficacy in extracting MalFGK2 into nanodiscs from proteoliposomes was characterized. The tested peptides were all modified with a Hex group at the N-tennini, and were designated as “F1W, F2W, F3W, and F4W.” In addition, the amphipathic repeat of SEQ ID NO: 1 was slightly increased to 20 or 22 amino acids (“20B and 22B”), resulting in stability enhancement of the nanodiscs. As compared to Hex- 18 A, several of the redesigned peptides showed improvement in extracting MalFGK2 into nanodiscs from proteoliposomes (FIGs. 10A-10B), with 20B exhibiting the highest efficacy. Next, Tip substitutions were placed in 20B, and performance was assessed for the reconstitution of MalFGKi nanodiscs from E. coli membranes. Consistent with the results obtained using proteoliposomes, Trp substituted 20B peptides were more effective than Hex-18A, giving rise to an approximately 2.5-fold increase in the yield of native nanodiscs. Because 20B 1 WA showed the highest reconstitution efficiency, native nanodiscs formed with this peptide were purified for further characterization. These native MalFGK2 nanodiscs formed by 20B1WA were quite homogenous as shown by negative stain EM (FIG. 4B) and contained native lipids including PC, PE, PG, and cardiolipin (FIG. 4D).

[0112] Since the peptide-encased nanodiscs retained native lipids, it was hypothesized that they might also be able to trap challenging membrane protein complexes that are otherwise disrupted by detergents. To test this hypothesis, a study was conducted to assess the performance of the peptides to isolate MalFGK2 mutants that usually fall apart once extracted out of membranes by detergents51. These mutants behave very differently from the wild-type protein and are useful tools for advancing the understanding of ABC transporters in general. However, biophysical dissection of these mutants is unattainable because they are unstable in detergents. As shown by native electrophoresis, several MalFGKi mutants were dissociated at various degrees after affinity purification in DDM (FIG. 4E, left). In contrast, 20B 1WA directly extracted many of these MalFGK2 mutants into nanodiscs with high efficiency and good stability for biochemical characterizations (FIG. 4E, right). The results showed that the basal ATPaseactivities of these mutants are much higher than the wild-type MalFGKi transporter (FIG. 4F), indicating escalated alternations in their conformational dynamics for MBP-indcpcndcnt maltose translocation.Example 6 - Reconstitution of eukaryotic membrane proteins into native nanodiscs using DeFrMSPs

[0113] To further interrogate the utility of DeFrMSPs for studies of membrane biology, an assay was carried out to assess their ability to reconstitute the adhesion GPCR mGluR7 expressed from HEK293T cells into native nanodiscs (FIG. 5A). mGluR7 was genetically fused with eGFP, allowing for sensitive fluorescent approaches to determine the efficiency of nanodisc formation (FIG. 11). Next, the collection of DeFrMSPs was incubated with mGluR7-eGFP membranes and the extracted samples were analyzed on native electrophoresis followed by ingel fluorescence imaging (FIG. 1 IB). The results showed that 20B 1WA was the most effective to extract mGluR7 into soluble nanodiscs as a single band of the reconstituted complex was observed on clear native PAGE (FIG. 5B and 1 IB). mGluR7 native nanodiscs formed by 20B1WA were purified by affinity purification and size-exclusion chromatography (SEC). Negative stain EM demonstrated that mGluR7 native nanodiscs are indeed quite monodisperse (FIG. 11C). Moreover, they are fully functional to bind with high affinity to the extracellular domain of their cognate interacting receptors Efln-1 and -2, as shown by native electrophoresis. See, e.g., FIG. 5C.

[0114] In addition to mGluR7, the performance of the engineered DeFrMSPs for native nanodisc reconstitution of the HCN channel was also tested (FIG. 6). Consistently, rHCNl was extracted into monodisperse nanodiscs from crude HEK293T membranes, as characterized by native electrophoresis, size-exclusion chromatography (SEC), and negative stain EM (data not shown). Interestingly, Hex-18A was the best scaffold for the extraction of HCN1 (data not shown), indicating that its local lipid environment might be different from mGluR7. Using a powerful single-molecule FRET assay (FIGs. 6B-6F), the result showed that HCN1 was functional in the peptide-encased native nanodiscs, retaining the activity to undergo cAMP- triggered conformational changes. The kinetics and affinity of HCNl-cAMP interactions in native nanodiscs are very different from data obtained in detergent micelles, supporting the roleof lipids in regulating the property of this ion channel. In contrast, it was not possible to efficiently extract them using amphipathic polymers, let alone maintain their activities.

[0115] Moreover, thin layer chromatography (TLC) experiments were performed to analyze the extracted lipids in native NDs (FIGs. 19E-19F). Interestingly, mGluR7 was associated with more PE lipids than HCN 1. This difference might suggest the role of lipids in regulating the function of membrane proteins. Further, these results demonstrate that DeFrMSP NDs can isolate surrounding native lipids with eukaryotic membrane proteins. Hence, the lipids extracted by DeFrNDs were analyzed. By performing lipidomic analysis, it was found that DeFrNDs did not show much preference for extracted lipids as compared to crude membranes (FIG. 20), suggesting that they can serve as useful tools to characterize the impact of native membranes on different kinds of membrane proteins.

[0116] Finally, it was assessed whether DeFrNDs are useful for isolating proteins embedded in ER and secretory vesicles. Sec61B from ER microsomes and VAMP2 from dense-core vesicles were well extracted by DeFrMSPs (FIG. 21).

[0117] The studies described herein encompass a wide range of protein families and establish that DeFrNDs are versatile tools for different kinds of membrane proteins resided in various cellular membranes.DISCUSSION

[0118] The present disclosure provides engineered peptides which allow detergent-free reconstitution of membrane proteins into nanodiscs from native membranes. The approach as described in the present disclosure bypasses the limitation of detergent-mediated reconstitution used in traditional methods, thereby enabling structural and functional studies of membrane protein complexes that may be previously unattainable. Moreover, the membranes in DeFrMSP nanodiscs can faithfully mimic native lipid environments for biophysical dissections of both integral and periphery membrane proteins involved in various transmembrane signaling pathways.

[0119] Without wishing to be bound by any particular theory, to obtain nanodiscs with cell membrane proteins, peptides need to penetrate lipid bilayers and then stably encircle a rigid patch of membranes into nanodisc structures. Although anti-bacterial peptides can robustlyinsert into membranes, they cannot provide the stability required to consistently generate nanodiscs. In contrast, nanodisc scaffold peptide (NSP)-bascd peptides arc capable of forming stable nanodisc structures. However, they are much less effective in penetrating the lipid bilayers to allow for detergent-free nanodisc reconstitution. Surprisingly, the peptides described herein (e.g., the peptides described herein having hexanoic acid modification) both efficiently insert into membranes and stably encircle membrane to form stable nanodiscs.

[0120] The DeFrMSPs described herein may extend the potential of nanodiscs for fundamental research of membrane biology. An advantage of the platform disclosed in the present disclosure is the ability to reconstitute asymmetric membranes with synthetic and native lipids. Such advantage may allow for further uses with a variety of biophysical approaches to investigate how lipid asymmetry regulates the conformational dynamics of membrane protein complexes as observed in cells. In addition, the size of DeFrNDs can be expanded to 30-50 nm when generated from protein-free liposomes (FIGs. 15A-15C), indicating that DeFrNDs can accommodate much larger membrane protein complexes than the ones assayed in the study described herein.

[0121] Another advantage of DeFrND is the ability to reconstitute asymmetric membranes with synthetic and native lipids. By virtue of this advantage, it is now possible to use a variety of biophysical approaches to investigate how lipid asymmetry regulates the conformational dynamics of membrane protein complexes as observed in cells. In conjunction with single particle cryoEM, the DeFrND technology is useful to unravel the molecular mechanism underlying the regulation of myriad transmembrane signaling pathways by native cell membranes. DeFrNDs have not exhibited preferred orientation as sometimes found in other membrane mimetic systems66. This advantage may provide a useful alternative option for structural characterizations of membrane proteins in a near native lipid environment using single particle cryoEM.

[0122] The peptides described herein may also benefit therapeutic candidates based on nanodisc delivery to host cells. Nanodiscs are very useful tools for vaccine development because of their nanoscale sizes, enhanced tissue penetration, and low immunogenicity. As such, these nanomaterials may avidly engage with immune cells and re-engineer the immune system to control cancer and viral infections. The enhanced stability and efficiency of detergent-freenanodisc reconstitution by the peptides of the disclosure are advantages for therapeutic application in these areas. Another application of the materials and methods described herein is the delivery of therapeutic protein complexes (e.g., genome editing reagents) to host cells. In conjunction with cationic cell penetrating peptides, amphipathic peptides can enclose protein therapeutics and facilitate their entry into the cells through the unconventional clathrin- independent endocytosis / macropinocytosis. The results described herein demonstrate that nanodiscs obtained as described herein are useful reagents for the delivery of therapeutic proteins.

[0123] Traditional membrane scaffold proteins derived from ApoAl are largely unable to disrupt membranes. While some studies on ApoAl mimetic peptides suggested that some of them can solubilize synthetic membranes with defined lipid compositions32,33, 37’64, the engineered peptides described herein can extract membrane protein-bearing discoids with high efficiency from native membranes. The reconstituted signaling membrane proteins (channels, receptors, and transporters) are never exposed to detergents throughout the reconstitution process, and the resultant nanodiscs maintain their structure, function and single molecule dynamics (FIGs. 2A-6B).

[0124] In addition, DeFrMSPs are compatible with various membrane proteins and vastly simplify the workflow for the reconstitution of nanodiscs compared to previous methods. Current native nanodiscs are formed using different kinds of polymers that often require careful optimization7, 27,such as the concentration of divalent ions and pH in the reconstitution buffer. The compatibility of DeFrNDs with bivalent cations makes it a great complimentary system to detergent-free reconstitution systems based on polymers. For example, DeFrNDs are suitable for membrane proteins that are not active in polymer-based NDs (FIGs. 1A-3D and FIGs. 12A-12F).

[0125] The instant disclosure establishes a new approach for native nanodisc reconstitution through the development of membrane scaffold peptides, DeFrMSPs.

[0126] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0127] As used herein, the singular' forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nanodisc" includes aplurality of nanodiscs and equivalents thereof known to those skilled in the art, and so forth. The term “about” signifies not more or less than 10 percent of the stipulated amount. Thus, a diameter of about 11 nm may be interpreted to be inclusive of 9.9 nm to 12.1 nm. It should be understood that, while various embodiments in the specification are presented using “comprising” language, under various circumstances, a related embodiment may also be described using “consisting of’ or “consisting essentially of’ language. The disclosure contemplates embodiments described as “comprising” a feature to include embodiments which “consist of’ or “consist essentially of” the feature. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0128] It should also be understood that when describing a range of values, the disclosure contemplates individual values found within the range. In any of the ranges described herein, the endpoints of the range are included in the range. The upper and lower limits may independently be included in smaller ranges within the range of values, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. The description also contemplates the same ranges in which the lower and / or the higher endpoint is excluded.

[0129] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several aspects. Any recited method can be carried out in the order of steps recited or in any other order which is logically possible. This is intended to provide support for all such combinations.

[0130] Preferred embodiments of this disclosure are described herein. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. This disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Also, only such limitations which are described herein as critical to theinvention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical arc intended as aspects of the invention.REFERENCES1. Bayburt, T.H., Grinkova, Y.V. & Sligar, S.G. Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins. 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Claims

CLAIMS1. A peptide comprising an amino acid sequence of formula I:DWX1KAFYDKX2AEKX3KEAX4 (I) (SEQ ID NO: 41) wherein:Xi is an amino acid with hydrophobic side chain;X2 is selected from V or W;X3 is selected from L or W;X4 is selected from F or W; and wherein the N-terminus of the peptide is modified with a fatty acid.

2. The peptide of claim 1, wherein the peptide comprises an amino acid sequence of formula II:DWX1KAFYDKX2AEKX3KEAX4X5W (II) (SEQ ID NO: 42) wherein X5 is selected from D or E.

3. The peptide of claim 1 or claim 2, wherein the peptide does not comprise SEQ ID NO: 1.

4. The peptide of any one of claims 1-3, wherein Xi is F, L, or W.

5. The peptide of any one of claims 2-4, wherein Xi is L, X2 is V, X3 is W, X4 is F, and X5 is D.

6. The peptide of any one of claims 1-5, wherein the peptide comprises an amino acid sequence of formula III:DWX1KAFYDKX2AEKX3KEAX4 X5WX6K (III) (SEQ ID NO: 43) wherein Xe is selected from L or F.

7. The peptide of any one of claims 1-6, which is amidated at the C-terminus.

8. The peptide of any one of claims 1-7, wherein the fatty acid is hexanoic acid.

9. The peptide of claim 1, comprising the amino acid sequence of any one of SEQ ID NOs: 22-40.

10. A method of preparing a nanodisc, the method comprising(a) contacting a lipid bilayer comprising a payload with a peptide comprising an amino acid sequence of formula I:DWX1KAFYDKX2AEKX3KEAX4 (I) (SEQ ID NO: 41) wherein:Xi is an amino acid with hydrophobic side chain;X2 is selected from V or W;X3 is selected from L or W;X4 is selected from F or W; wherein the N-terminus of the peptide is modified with a fatty acid; and(b) purifying a nanodisc comprising the payload, wherein the method does not comprise a reconstitution step using detergent.

11. The method of claim 10, wherein the peptide comprises an amino acid sequence of formula II:DWX1KAFYDKX2AEKX3KEAX4 X5W (II) (SEQ ID NO: 42) wherein X5 is selected from D or E.

12. The method of claim 10 or claim 11, wherein the peptide does not comprise SEQ ID NO: 1.

13. The method of any one of claims 1-12, wherein the fatty acid is hexanoic acid.

14. The method of any one of claims 1-12, wherein the fatty acid comprises a myristoyl moiety, decanoic acid, or palmitoyl moiety.

15. The method of any one of claims 10-14, wherein the peptide is amidated at the C-tcrminus.

16. The method of claim 10, wherein the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 22-40.

17. The method of claim 10, wherein the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 7-21.

18. The method of any one of claims 10-17, wherein the lipid bilayer is a cell membrane fraction.

19. The method of any one of claims 10-18, wherein the payload is a protein.

20. The method of any one of claims 10-18, wherein the payload is a transporter protein, a receptor, or an ion channel.

21. The method of any one of claims 10-20, wherein the payload is a detergent sensitive protein.

22. The method of any one of claims 10-21, wherein the lipid bilayer is an asymmetric membrane and the nanodisc retains membrane asymmetry.

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