Synergy between desiccation protective solutes and disordered proteins

Abstract

Embodiments of the present disclosure generally relate to compositions, methods, and systems for desiccation tolerance. Embodiments described herein also generally relate to compositions and methods for stabilizing a biological material. In an embodiment is provided a method that includes introducing an exogenous cosolute with an intrinsically disordered protein to induce aggregation or oligomerization of the intrinsically disordered protein.

Description

SYNERGY BETWEEN DESICCATION PROTECTIVE SOLUTES AND DISORDERED PROTEINSGOVERNMENT RIGHTS

[0001] This invention was made with United States government support under Grant No. 2128069 awarded by the National Science Foundation. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0002] This application contains references to amino acid and nucleic acid sequences which have been submitted as the sequence listing text file entitled “SEQ ID NOS 1-20”, file size 22.3 Kilobytes (KB), created March 11, 2025, which is hereby incorporated by reference into this application in its entirety.CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of and priority to U.S. Provisional Patent Application No. 63 / 569,676, filed on March 25, 2024, which is incorporated herein by reference in its entirety.FIELD

[0004] Embodiments described herein generally relate to compositions and methods for stabilizing a biological material or biologically-derived material. Embodiments of the present disclosure generally relate to compositions, methods, and systems for desiccation tolerance.BACKGROUND

[0005] Intrinsically disordered proteins (IDPs) make up about 40% of the eukaryotic proteome. Unlike typical well-folded proteins, IDPs are characterized by a lack of defined tertiary structure, and instead exist as an ensemble of dynamic, interconverting conformations. Despite their disordered nature, IDPs play important roles in many biological processes including regulation of transcription and translation, metabolic signaling, subcellular organization, molecular chaperoning and response and adaptation to environmental cues. There is little understanding into how IDPs and cosolutes provide desiccation protection. In addition, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance.

[0006] There is a need for a new and improved understanding of desiccation tolerance. There is also need for new compositions and methods for stabilizing a biological material or a biologically-derived material.SUMMARY

[0007] Embodiments described herein generally relate to compositions and methods for stabilizing a biological material or biologically-derived material. Embodiments of the present disclosure generally relate to compositions, methods, and systems for desiccation tolerance. The inventor found that the protective function of desiccation-protective intrinsically disordered proteins (IDPs) may be enhanced by, for example, conformational changes in the IDP induced by cosolutes. Results described herein demonstrate that functional synergy between IDPs and cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet, the mechanisms underlying this synergy differ between IDP families.

[0008] In an embodiment is provided a method that includes introducing an exogenous cosolute with an intrinsically disordered protein (IDP) to induce aggregation or oligomerization of the IDP, the IDP comprising an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof.

[0009] In another embodiment, a method of inducing aggregation or oligomerization of a protein is provided. The method includes introducing an exogenous disaccharide with a monomeric intrinsically disordered protein (IDP) to form a composition comprising the exogenous disaccharide and an aggregated form or oligomerized form of the IDP, the monomeric form of the IDP comprising an amino acid sequence having at least 90% identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof, a molar ratio of the exogenous disaccharide to the IDP is from about 10: 1 to about 1,000: 1.

[0010] In another embodiment is provided a composition that includes an oligomeric form of an IDP, the IDP comprising an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof; and an exogenous cosolute.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] So that the manner in which the above recited features of the present disclosure may be understood in detail, a more particular description of the disclosure,briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only exemplary embodiments and are therefore not to be considered limiting of its scope, and may admit to other equally effective embodiments.

[0012] FIGS. 1 A and IB show non-limiting data indicating that LEA motifs are not synergistic with cosolutes: FIG. 1A) Synergy plots for trehalose:LEA peptide at 100: 1 molar ratio. n=3, Welch’s t-test was used for statistical comparison, error bars = standard deviation. FIG. IB) Synergy plots for sucrose:LEA peptide at 100: 1 molar ratio. Disaccharide (hatched); LEA peptide (different hatching); combined (no hatching).

[0013] FIG. 2 shows non-limiting data indicating that cosolutes increase the global dimensions of CAHS D (0.156 mM). Analyzed data from high-throughput SAXS experiments of protein at 4 mg / mL in 20 mM tris HC1 (pH 7.00). The left plot shows the radius of gyration of the protein in the presence of no cosolute, molar ratios of 1.6: 1 trehalose:CAHS D, 16: 1 trehalose:CAHS D, 160: 1 trehalose:CAHS D, 1.6: 1 sucrose:CAHS D, 16:1 sucrose:CAHS D, and 160: 1 sucrose:CAHS D. Error bars represent uncertainty in the measurement, provided by BioXTAS RAW. The right plot shows molecular weight values derived from Guinier analysis using a concentrationindependent Bayesian assessment. 160: 1 trehalose:CAHSD and 160: 1 sucrose:CAHS D show a higher pMW indicating the higher order assembly of CAHS D. The dashed horizontal line (right panel) indicates the monomeric protein’s molecular weight. Error bars represent >90% confidence interval, which is directly obtained from the Bayesian method.

[0014] FIGS. 3A and 3B show non-limiting data indicating that cosolutes promote gelation of CAHS D at non-gelling concentrations. FIG. 3 A) DSC thermogram of 0.235 mM CAHS D protein with trehalose and sucrose at molar ratios of 1 : 1, 10: 1, 100: 1, and 500: 1 (cosolute: CAHS D protein). FIG. 3B) Change in enthalpy measurements for cosolute:CAHS D protein mixtures relative to CAHS D protein. Enthalpy measurements were done by taking the area of gel melt peaks represented by black vertical dashes in FIG. 3 A (see methods).

[0015] FIGS. 4A, 4B, 4C, and 4D show non-limiting data indicating that transfer free energy of CAHS D highlights the difference between synergistic and non- synergistic cosolutes. FIG. 4 A) Change in enthalpy measurements for betaine: CAHS Dmixtures relative to CAHS D at 6 mg / mL in varying molar ratios of glycine betaine:CAHS D (0: 1, 1 : 1, 10: 1, 100: 1, and 500: 1). Enthalpy measurements were done by taking the area of gel melt peaks. FIG. 4B) DSC thermograms of CAHS D at 12 mg / mL (0.47 mM) in varying molar ratios of glycine betaine:CAHS D (0: 1, 1 : 1, 10: 1, 100: 1, and 500: 1). FIG. 4C) Lactate Dehydrogenase (LDH) synergy assay for glycine betaine and CAHS D at various molar ratios. FIG. 4D) A scatter plot depicting the relationship between the AAG^Dof a given cosolute with CAHS D and its synergy in the LDH assay at different molar ratios, p-value is given from a Pearson correlation.

[0016] FIGS. 5A-5C show non-limiting synergy plots for various cosolutes with FL-proline as a function of molar ratio (cosolute:FL-proline). FIG. 5A) data for trehalose :FL-proline. FIG. 5B) data for sucrose:FL-proline. FIG. 5C) shows data for betaine:FL-proline.

[0017] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.DETAILED DESCRIPTION

[0018] Embodiments described herein generally relate to compositions and methods for stabilizing a biological material or biologically-derived material. Embodiments of the present disclosure generally relate to compositions, methods, and systems for desiccation tolerance.

[0019] The conformational ensemble and function of IDPs are sensitive to their solution environment. The inherent malleability of disordered proteins combined with the exposure of their residues accounts for this sensitivity. One context in which IDPs play roles that are concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has been linked to the enrichment of IDPs such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat soluble (CAHS) proteins. Despite knowing that IDPs play roles during desiccation, little is known mechanistically about how IDP- cosolute interactions influence desiccation tolerance. Here, the inventor tested the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by cosolutes. The inventor found that desiccation- related IDPs derived from four different organisms and spanning two LEA proteinfamilies and the CAHS protein family, synergize best with cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. The inventor further demonstrated that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel under the conditions tested. Results provided herein demonstrate that functional synergy between IDPs and cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet, the mechanisms underlying this synergy differ between IDP families.

[0020] IDPs make up about 40% of the eukaryotic proteome. Unlike typical well- folded proteins, IDPs are characterized by a lack of defined tertiary structure, and instead exist as an ensemble of dynamic, interconverting conformations. Despite their disordered nature, IDPs play roles in many biological processes including regulation of transcription and translation, metabolic signaling, subcellular organization, molecular chaperoning and response and adaptation to environmental cues. Despite lacking a stable three-dimensional structure, IDPs still follow a similar paradigm by which form begets function. Different from well-folded proteins, however, an IDP’s sequence determines the ensemble of conformations it adopts, and this ensemble may be important for the IDP’s function(s). However, sequence is not the only determinant of the conformations present in an IDP’s ensemble. This is because IDP ensembles have relatively few intramolecular bonds and a large solvent-accessible surface area, which makes their ensembles more sensitive to the physicochemical environment than the relatively rigid structures of well-folded proteins.

[0021] The sensitivity of IDP ensembles to their solution environment and their link to IDP function poses a fundamental question: how do sequence and solution combine to tune IDP ensemble and function? To explore this question, the inventor explored a biological phenomenon where the intracellular environment undergoes drastic physical chemical changes: desiccation.

[0022] Organisms across every biological kingdom can survive near-complete desiccation by entering a state of reversible suspended metabolism known as anhydrobiosis (from Greek for ‘life without water’). As water effluxes from the cell during drying, the concentration of cosolutes increases by orders of magnitude, dramatically changing the phy si cochemistry of the cell. In addition to the decrease in water content and concomitant increase in cosolute concentrations, the composition ofthe intracellular environment changes massively because of a regulated metabolomic response to drying mounted by anhydrobiotic organisms.

[0023] The acquisition of desiccation tolerance has historically been linked to the intracellular buildup of cosolutes such as trehalose, sucrose, arabinose, stachyose, and raffinose in plants and trehalose in some animals, fungi, and bacteria. More recently, the accumulation of high levels of IDPs has also been linked to desiccation tolerance in many organisms. Common examples of desiccation-related IDPs include LEA proteins. LEA proteins are classified into seven different families based on the presence of conserved motif sequences. Another family of desiccation-related IDPs are tardigrade- specific CAHS proteins.

[0024] To reveal the underpinnings of cosolute-IDP synergy, the inventor examined the secondary and tertiary structure of protective IDPs in the presence of two disaccharides that are similar in terms of size but distinct with respect to chemistry and use across taxa. In all cases, the secondary structure (residual helicity) and tertiary structure (radius of gyration) do not change significantly in the presence of synergistic cosolutes and thus cannot explain the enhancement in function observed with synergistic cosolutes. Next assessed were quaternary structure as both CAHS and LEA proteins oligomerize, and for CAHS proteins, oligomerization leads to gelation. While synergistic cosolutes did not influence LEA oligomerization, CAHS D oligomerization and gelation were enhanced in the presence of synergistic cosolutes. The inventor further show that CAHS D’s synergy may be explained through direct repulsive interactions between cosolutes and CAHS D’s sidechains. However, this explanation does not hold for LEA proteins, implying that synergy in different protein families occurs through distinct mechanisms.

[0025] Investigations provided herein indicate that different families of protective IDPs may have orthogonal modes of action and different functions in divergent solution environments. Beyond expanding the understanding of desiccation tolerance, these findings shed light on the sensitivity of IDP ensemble and function to the chemical composition of their environment. This is important as IDPs are ubiquitous across biology and function in key developmental processes and disease states that are concomitant with large changes in intracellular chemistry. Understanding how disordered proteins interact and evolve with the solution environment will provide insights into these biological mechanisms and phenomena.

[0026] The use of headings is for purposes of convenience only and does not limit the scope of the present disclosure. Embodiments described herein may be combined with other embodiments.

[0027] As used herein, a “composition” may include component(s) of the composition, reaction product(s) of two or more components of the composition, a remainder balance of remaining starting component(s), or combinations thereof.

[0028] Embodiments described herein generally relate to compositions and methods for stabilizing a biological material of interest present in the composition in a dry state and / or at temperatures of, for example, ambient temperature or higher.

[0029] Compositions described herein may include a biological material of interest, an IDP, an optional exogenous cosolute, and optionally water. These compositions may be characterized as protecting and / or stabilizing the biological material of interest present in the composition in a dry state at a temperature of ambient temperature or higher (e.g., about ambient temperature to less than about 100°C, such as a temperature of room temperature or higher). Ambient temperature refers to 20°C.

[0030] The term “dry state”, when referring to a composition described herein, refers to a composition that has about 15 wt% or less water based on the total wt% of the composition. In some examples, a composition in a dry state has an amount of water in a range from about 0 wt% or more, about 15 wt% or less, or combinations thereof, such as from greater than 0 wt% to about 15 wt%, from about 2 wt% to about 12 wt%, from about 5 wt% to about 15 wt%, from about 6 wt% to about 14 wt%, such as from about 7 wt% to about 12 wt%, such as from about 8 wt% to about 10 wt%, or from about 10 wt% to about 14 wt% based on a total wt% of the composition. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range.

[0031] The biological material of interest is different from the IDP. The biological material of interest may be in its natural state, in a modified state, derived from a living organism, and / or synthesized. The biological material of interest may include a peptide, a polypeptide, a protein, an enzyme, an antibody, a globular protein, a hormone, a natural product, a derivative thereof, a component thereof, or combinations thereof, among others. The biological material of interest may include a biologically-derived material of interest. A biologically-derived material of interest may be derived from a peptide, a polypeptide, a protein, an enzyme, an antibody, a globular protein, ahormone, a natural product, a derivative thereof, a component thereof, or combinations thereof, among others. The biological material of interest and the biologically-derived material of interest is not an IDP and is not derived from an IDP. The biological material of interest or the biologically-derived material of interest may be a pharmaceutical.

[0032] Any suitable optional exogenous cosolute may be utilized. For example, the optional exogenous cosolute may include a disaccharide. Illustrative, but non-limiting, disaccharides may include trehalose, sucrose, maltose, lactose, or combinations thereof, such as sucrose, trehalose, or combinations thereof. More than one exogenous cosolute may be utilized in compositions described herein.

[0033] The IDP may include a protein or polypeptide (which may be isolated) comprising, consisting essentially of, or consisting of:

[0034] (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 20, or a complement thereof;

[0035] (b) an amino acid sequence encoded by a nucleotide sequence of any one ofSEQ ID NOs: 14, 15, 16, 17, 18, 19, or a complement thereof;

[0036] (c) an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to one or more of SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof;

[0037] (d) an amino acid sequence encoded by a nucleotide sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to one or more of SEQ ID Nos: 14, 15, 16, 17, 18, 19, or a complement thereof; and / or

[0038] (e) a functional fragment of any one of (a) to (d).

[0039] More than one IDP may be utilized with compositions of the present disclosure.

[0040] Polypeptides (proteins) and fragments thereof may be modified for use by the addition, at the amino- and / or carboxyl-terminal ends, of a blocking agent. Such blocking agents may include, for example, additional related or unrelated peptide sequences that may be attached to the amino and / or carboxyl terminal residues of the peptide to be administered. For example, one or more non-naturally occurring aminoacids, such as D-alanine, may be added to the termini. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art may be attached to the amino and / or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus may be replaced with a different moiety. Additionally, the peptide terminus may be modified, for example, by acetylation of the N-terminus and / or amidation of the C-terminus. Likewise, the peptides may be covalently or noncovalently coupled to pharmaceutically acceptable “carrier” proteins prior to use.

[0041] The exogenous cosolute may be present in the composition in any suitable amount. For example, a molar ratio of the exogenous cosolute(s) to the IDP(s) in compositions described herein may be in a range from about 1 : 100 to about 100: 1, such as from about 1 : 10 to about 10: 1, such as about 1 : 1 (exogenous cosoluteTDP). A molar ratio of the exogenous cosolute(s) to the IDP(s) in compositions described herein may be greater than about 10: 1, such as in a range from about 10: 1 to about 1,000: 1, such as from about 50: 1 to about 1,000: 1, such as from about 100:1 to about 500: 1, such as from about 1 :200 to about 1 :400 (exogenous cosolute :IDP). Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range.

[0042] The exogenous cosolute may be present in the composition in an amount sufficient to induce gelation of the IDP present in the composition.

[0043] Compositions described herein may further include water. For example, the composition may include 15 wt% or less of water based on a total wt% of the composition. The composition has an amount of water that may be from about 0 wt% or more, about 15 wt% or less, or combinations thereof, such as from greater than 0 wt% to about 15 wt%, or from about 2 wt% to about 12 wt%, or from about 5 wt% to about 15 wt%, or from about 6 wt% to about 14 wt%, or from about 7 wt% to about 12 wt%, or from about 8 wt% to about 10 wt%, or from about 10 wt% to about 14 wt%, or from about 5 wt% to about 12 wt% based on a total wt% of the composition. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range.

[0044] In some embodiments, which may be combined with other embodiments, a composition may include a molar ratio of the biological material (and / or biologically- derived material) of interest to the exogenous cosolute that may be in a range fromabout 1 :0.1 to about 1 : 100 (biological material : exogenous cosolute), such as from about 1 :0.5 to about 1 :50, such as from about 1 :2.5 to about 1 :25, such as from about 1 :5 to about 1 :15, such as about 1 : 10. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range. The composition may include other components described herein.

[0045] For example, a composition may include a molar ratio of component A:component B, where component A refers to a biological material of interest and / or biologically-derived material of interest, and component B refers to the exogenous cosolute. For example, a composition may include a LDH: exogenous cosolute molar ratio of about 1 : 10.

[0046] The total wt% of the composition may be based on the wt% of the biological material (and / or biologically-derived material) of interest, plus the wt% of the IDP, plus the wt% of the optional exogenous cosolute, plus the wt% of the optional water. The total wt% of the composition does not exceed 100 wt%.

[0047] Additionally provided herein is a recombinant nucleic acid construct comprising, consisting essentially of, or consisting of:

[0048] (a) a nucleotide sequence of any one of SEQ ID NOs: 14, 15, 16, 17, 18, 19, or a complement thereof;

[0049] (b) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof;

[0050] (c) a nucleotide sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to a nucleotide sequence of any one of (a) or (b);

[0051] (d) a nucleotide sequence which anneals under stringent hybridization conditions to the nucleotide sequence of any one of (a) to (c), or a complement thereof;

[0052] (e) a nucleotide sequence that differs from the nucleotide sequences of any one of (a) to (d) above due to the degeneracy of the genetic code;

[0053] (f) a functional fragment of a nucleotide sequence of any one of (a) to (e); or

[0054] (g) any combination of the nucleotide sequences of (a)-(f).

[0055] In some embodiments, the nucleic acid, nucleotide sequence, or polynucleotide described herein may be a complement (which may be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide of the present disclosure. Two nucleotide sequences may be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. In some embodiments, two nucleotide sequences considered to be substantially complementary hybridize to each other under highly stringent conditions. Stringent conditions refers to a melting temperature above 65°C, indicating the strength of the hybridization.

[0056] The nucleotide sequences and / or recombinant nucleic acid molecules of the present disclosure may be operatively linked and / or associated with a variety of promoters for expression in cells. Thus, a recombinant nucleic acid described herein may further include one or more promoters operably linked to one or more nucleotide sequences.

[0057] The recombinant nucleic acid molecule may be an expression cassette or may be included within an expression cassette. As used herein, “expression cassette” refers to a recombinant nucleic acid molecule comprising a nucleotide sequence of interest (for example, a nucleotide sequence encoding an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof; and / or a nucleotide sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, about at least 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 14, 15, 16, 17, 18, 19, or a complement thereof, wherein said nucleotide sequence may be operably associated with at least a control sequence (for example, a promoter). Accordingly, some embodiments of the present disclosure provide expression cassettes designed to express the nucleotide sequences described herein in a cell.

[0058] An expression cassette comprising a nucleotide sequence may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. An expression cassette may be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Anexpression cassette may optionally include a transcriptional and / or translational termination region (i.e., termination region) that is functional in the cell in which the nucleotide sequence of interest is to be expressed.

[0059] A variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation. The termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host organism, or may be derived from another source (i.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, the host organism, or any combination thereof). In addition, a coding sequence’s native transcription terminator may be used.

[0060] An expression cassette may include a nucleotide sequence for a selectable marker, which may be used to select a transformed organism and / or cell. As used herein, “selectable marker” refers to a nucleotide sequence that when expressed imparts a distinct phenotype to the transformed organism or cell expressing the marker and thus allows such transformed organisms or cells to be distinguished from those that do not have the marker. Such a nucleotide sequence may encode a selectable or screenable marker, depending on whether the marker confers a trait that may be selected for by chemical means, such as by using a selective agent (for example, an antibiotic, herbicide, or the like), or on whether the marker is simply a trait that one may identify through observation or testing, such as by screening. Of course, many examples of suitable selectable markers useful in various organisms are known in the art and may be used in the expression cassettes described herein.

[0061] In addition to expression cassettes, the nucleic acid molecules and nucleotide sequences described herein may be used in connection with vectors. The term “vector” refers to a composition for transferring, delivering, or introducing a nucleic acid (or nucleic acids) into a cell. A vector includes a nucleic acid molecule comprising the nucleotide sequence(s) to be transferred, delivered, and / or introduced. Vectors for use in transformation of animals, plants, and other organisms are well known in the art. Illustrative, but non-limiting, examples of general classes of vectors include a viral vector, a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a fosmid vector, a bacteriophage, an artificial chromosome, or an agrobacterium binary vector in double or single stranded linear or circular form which may or may notbe self-transmissible or mobilizable. A vector may transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (for example, an autonomous replicating plasmid with an origin of replication). Additionally included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from prokaryotic and eukaryotic organisms. In some embodiments, the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, for example, bacterial, or an animal or a plant cell. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.

[0062] Embodiments of the present disclosure also generally relate to methods of forming the compositions, methods of stabilizing a biological material of interest and / or biologically-derived material of interest, and to methods of forming a stabilized biological material of interest and / or a stabilized biologically-derived material of interest. Embodiments described herein may be used to stabilize the least one biological material of interest (and / or biologically-derived material of interest) present in the composition at temperatures of, for example, ambient temperature or higher.

[0063] As described herein, the inventor found that contact of an IDP monomer with an exogenous cosolute may induce oligomerization and / or aggregation of IDP monomers. The oligomers (or oligomeric form of the IDP) may be dimers, trimers, tetramers, etc. The oligomers may arise from non-covalent interactions such as electrostatic, hydrophobic, and / or hydrogen bonding. While not wishing to be bound by any theory, it is believed that all three of these non-covalent interactions may drive oligomerization.

[0064] In at least one embodiment, which may be combined with other embodiments, methods described herein may generally include introducing or contacting the biological material of interest and / or biologically-derived material of interest with the IDP (in monomeric or oligomeric form) and the optional exogenous cosolute to form the composition comprising the biological material of interest and / or biologically-derived material of interest, the IDP, and the exogenous optional cosolute,thereby, for example, stabilizing the biological material of interest and / or biologically- derived material of interest.

[0065] Introducing or contacting may be performed under conditions that include suitable temperatures, pressures, and rates of introduction of the biological material of interest and / or biologically-derived material of interest with the IDP and the optional exogenous cosolute. The conditions may also include mixing the components of the composition by any suitable mixing process.

[0066] In some embodiments, which may be combined with other embodiments, methods described herein may include introducing or contacting an IDP with an exogenous cosolute, and then the resulting mixture may be introduced or contacted with the biological material of interest and / or biologically-derived material of interest.

[0067] Prior to forming the composition, the components (e.g., the biological material of interest and / or biologically-derived material of interest, the IDP, and the optional exogenous cosolute) may be present in an aqueous material such as water or an aqueous buffer. The aqueous buffer may be any suitable aqueous buffer such as Tris or PBS. The components may be mixed.

[0068] The method may further include removing at least a portion of the liquid content of the composition. The liquid content may include the aqueous material. Removing at least a portion of the liquid content may include drying, or at least partially drying, the composition that includes the biological material of interest and / or biologically-derived material of interest, the IDP, and the optional exogenous cosolute. Any suitable method of drying may be utilized such as, for example, air drying, evaporating (e.g., with a hot plate), dehydrating, desiccating, vacuum desiccating, vacuum drying, spray drying, freeze drying, spray-freeze drying, lyophilizing, foam drying, or combinations thereof, among other suitable methods.

[0069] The drying or at least partially drying provides the composition in the dry state, e.g., having a water content in a range from 0 wt% to about 15 wt% as described above.

[0070] “Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes (i) an mRNA that is translated into an amino acid sequence of a protein; or (ii) a functional RNA, such as an interfering RNA or antisense molecule.

[0071] “Recombinant,” when used with reference to, e.g., a cell, nucleic acid, polypeptide, expression cassette or vector, refers to a material, or a materialcorresponding to the natural or native form of the material, that has been modified by the introduction of a new moiety or alteration of an existing moiety, or is identical thereto but produced or derived from synthetic materials. For example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell (i.e., “exogenous nucleic acids”) or express native genes that are otherwise expressed at a different level, typically, under-expressed or not expressed at all.

[0072] Recombinant techniques may include, e.g., use of a recombinant nucleic acid such as a cDNA encoding a protein or an antisense sequence, for insertion into an expression system, such as an expression vector; the resultant construct is introduced into a cell, and the cell expresses the nucleic acid, and the protein, if appropriate. Recombinant techniques also encompass the ligation of nucleic acids to coding or promoter sequences from different sources into one expression cassette or vector for expression of a fusion protein, constitutive expression of a protein, or inducible expression of a protein.

[0073] The term “nucleic acid” may be in the form of RNA or in the form of DNA, and include messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA. The DNA may be double-stranded or single-stranded, and if single-stranded may be the coding strand or the non-coding (anti-sense, complementary) strand.

[0074] As used herein, a “variant” is a nucleic acid, protein or polypeptide which is not identical to, but has significant homology (for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) over the entire length of the wild type nucleic acid or amino acid sequence, as exemplified by sequences in the public sequence databases, such as GenBank. As used herein, a “protein, polypeptide or peptide fragment thereof’ refers to the full-length protein or a portion of it having an amino acid sequence usually at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in length, although dipeptides, tripeptides and tetrapeptides are also contemplated and encompassed by the present disclosure.

[0075] As used herein, a “mutant” is a mutated protein designed or engineered to alter properties or functions relating to glycosylation, protein stabilization, and / or ligand binding.

[0076] As used herein, the terms “native” or “wild-type” relative to a given cell, polypeptide, nucleic acid, trait or phenotype, refers to the form in which that is typically found in nature.

[0077] As used herein, the terms “protein,” “polypeptide,” “oligopeptide” and “peptide” have their conventional meaning and are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Furthermore, the polypeptides described herein are not limited to a specific length. Included within this definition are D- and L- amino acids, and mixtures of D- and L-amino acids. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Polypeptides may also refer to amino acid subsequences comprising epitopes, i.e., antigenic determinants substantially responsible for the immunogenic properties of a polypeptide and being capable of evoking an immune response.

[0078] “Position corresponding to” refers to a position of interest (i.e., base number or residue number) in a nucleic acid molecule or protein relative to the position in another reference nucleic acid molecule or protein. Corresponding positions may be determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues, for example, such that identity between the sequences is greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99%. The position of interest is then given the number assigned in the reference nucleic acid molecule. For example, if a particular polymorphism in Gene-X occurs at nucleotide 173 of SEQ ID No. X, to identify the corresponding nucleotide in another allele or isolate, the sequences are aligned and then the position that lines up with 173 is identified. Because various alleles may be of different length, the position designate 173 may not be nucleotide 173, but instead is at a position that “corresponds” to the position in the reference sequence.

[0079] “Percentage of sequence identity” and “percent (%) identity” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions)for alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Alignment of sequences for comparison may be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. AppL Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'L Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as “seeds” for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score may be increased. Cumulative scores are calculated using,for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0080] While all of the above mentioned algorithms and programs are suitable for a determination of sequence alignment and % sequence identity, for purposes of the disclosure herein, determination of % sequence identity will typically be performed using the NCBI BLAST, using default parameters provided.

[0081] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure, and are not intended to limit the scope of embodiments of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used but some experimental errors and deviations should be accounted for.Examples1. Materials, Experiments, and MethodsLA. Cloning

[0082] Inserts for example full-length proteins — AavLEAl, AtLEA4-2, AvLEAlC, CAHS D, and HeLEA68614 — were synthesized as codon optimized gBlocks (Integrated DNA Technologies) and cloned into the pET28b expression vector using gibson assembly (New England Biosciences). AtLEA3-3 was cloned in pET28a vector by Twist Bioscience. Clones were propagated in DH5a cells (NEB, Cat. #C2987H) and verified by Sanger sequencing (Eton Bioscience). l.B. Protein Expression

[0083] Expression constructs were transformed into BL21 (DE3) cells (New England Biosciences, Cat. #C2527H) and plated on Luria-Bertani (LB) agar plates with 50 pg / mL kanamycin. At least 3 single colonies were chosen for each construct and tested for expression. Constructs were expressed in 1 L LB / kanamycin medium and grown at 37°C while shaking at 180 rpm (Eppendorf Innova S44i) until an OD600 of 0.6 was reached. The culture was induced with 1 mM IPTG, and grown for the next 4 hours while shaking. AvLEAlC was grown for 1 hour following IPTG addition. Cells were harvested by centrifugation at 4000 rpm for 30 minutes at 4°C. Cell pellets were resuspended in 5 mL of 20 mM Tris buffer, pH 7.5 supplemented with 30 pL of IX protease inhibitor [Sigma Aldrich, Cat. P2714]). Cell pellets were stored at -80°C until further use. l.C. Protein Purification

[0084] Frozen pellets were thawed at room temperature, subjected to heat lysis in boiling water for 10 minutes, and cooled down for 15 minutes. These were then centrifuged at 10500 rpm at 10°C for 30 minutes, and the supernatant was later filter- sterilized through a 0.22 pm filter to remove any insoluble particles (EZFlow Syringe Filter, Cat. 388-3416-OEM). The filtrate was diluted two times the volume with buffer UA (8 M urea [Acros Organics, CAS No. 57-13-6], 50 mM sodium acetate [Tocris CAS No. 127-09-3], pH 4). This was loaded onto a HiPrep SP HP 16 / 10 (Cytiva, Cat. 29018183) cation exchange column and purified on an AKTA Pure (Cytiva, Cat. #29018224), controlled using the UNICORN 7-9.1 Workstation pure-BP-exp (Cytiva, Cat. #29128116). CAHS D was eluted using a 0-40% UB (8 M urea, 50 mM sodium acetate, and 1 M NaCl, pH 4) gradient and fractionated over 15 column volumes. LEA proteins were eluted using the 0-70% UB gradient over 15 column volumes. Protein fractions were assessed using SDS-PAGE and selected fractions were dialyzed in a 3.5 kDa tubing (SpectraPor 3 Dialysis Membrane, Part No. 132724) in 20 mM sodium phosphate buffer pH 7, followed by six rounds of Milli-Q water (18.2 MQcm) at four hours interval each. Concentration of the dialyzed fractions were then quantified using Qubit 4 fluorometer (Invitrogen, REF Q33226), flash frozen, then lyophilized (Labconco FreeZone 6, Cat. 7752021) for 48 hours, and stored at -20°C until further use. l.D. LEA Motif Sequence Identification

[0085] LEA 4 and LEA l sequence motifs were identified in full-length LEA proteins using RADAR (https: / / www.ebi.ac.uk / Tools / pfa / radar / ). In cases where RADAR was unable to identify repetitive motifs (e.g., in cases where a full-length LEA protein had only one or two motif repeats), manual selection and alignment of motifs was performed. l.E. Lactate Dehydrogenase (LDH) Protection Assay

[0086] An LDH assay was adopted from Nguyen K et al, Commun Biol, 2022, 5: 1046; Boothby TC et al., Mol Cell, 2017, 65: 975-984. e5; Goyal K et al., Biochem J, 2005, 388: 151-157; and Piszkiewicz S et al., Protein Sci, 2019, 28: 941-951. Proteins and individual additives were resuspended at a final concentration range 20 mg / mL to 0.1 mg / mL in 25 mM Tris HC1 pH 7. Rabbit muscle L-lactate dehydrogenase, sourced from Sigma (Sigma-Aldrich, Cat 10127230001), was added to each solution at a concentration of 0.1 mg / mL. Half of this sample was dried in a vacuum desiccator (SAVANT Speed Vac Concentrator) for 16 hours, while the other half was refrigerated at 4°C for the same duration. Water was added to both desiccated and non-desiccated samples to a final volume of 250 pL each. 10 pL sample was mixed with 980 pL phosphate pyruvate buffer (100 mM sodium phosphate, 2 mM sodium pyruvate; pH 6.00) supplemented with 10 pL of 10 mM NADH (Sigma-Aldrich NADH; disodium salt, grade II) in a quartz cuvette. LDH activity was measured as the kinetics of the decrease in NADH absorption at 340 nm for a minute in NanodropOne (Thermo Scientific). Percent protection was calculated as a ratio of NADH absorbance for the desiccated samples normalized to non-desiccated controls. Each sample was performed in triplicate. l.F. Lactate Dehydrogenase (LDH) Synergy Assay

[0087] The protection data for individual protein or motif was used to select a protective concentration. Trehalose or sucrose was mixed in equal parts with proteins at 2X concentration in 100 pL resuspension buffer (25 mM Tris HC1 pH 7) at respective molar ratios. LDHs assay was performed for the mixtures as described previously. For each mixture, LDH protection was assessed individually and as a mixture. The sum of the protection conferred by individual protein and cosolute was determined, which would refer to the expected additive protection. Synergy was determined by statistical comparison of this expected protection with the experimental protection. l.G. Circular Dichroism (CD) Spectroscopy

[0088] CD spectroscopy was adopted from Bremer A. et al. “Folding of intrinsically disordered plant LEA proteins is driven by glycerol-induced crowding and the presence of membranes,” FEBS J, 2017, 284: 919-936. Lyophilized proteins were resuspended in 25 mM NaPi pH 7 to a concentration of 200 pM. The resuspended protein was then mixed in equal parts with the NaPi buffer, 20 mM trehalose, or 20 mM sucrose in separate samples to a 100: 1 molar ratio, with a final protein concentration of 100 pM and cosolute concentration of 10 mM. Protein concentration was confirmed with either a UV-vis (Thermo Scientific, GENESYS 50 UV-visible spectrophotometer) or a Qubit (Life Technologies, Qubit 3.0 Fluorometer). 20 pL aliquots of the samples were deposited on a 0.05 mm quartz cuvette and measured in a Circular Dichroism (CD) spectrometer (JASCO, J- 1500 model). New 20 pL aliquots were then deposited on one half of a 0.05 mm quartz cuvette and spread across part of the cuvette with the tip of a pipette, to a surface area of about 1 cm2. The samples were then desiccated in a vacuum chamber with drierite for 1 hour to create a dry film, and another CD measurement was taken immediately after the vacuum was stopped. Each sample was performed in triplicate. l.H. Small-Angle X-ray Scattering (SAXS) - Sample Preparation

[0089] Lyophilized protein was resuspended at high concentration in a buffer containing 20 mM tris HC1 (pH = 7.0) and the correct amount of cosolute to reach the desired molar ratio. Protein samples were then quantified with the Qubit Protein Assay from ThermoFisher Scientific (catalog# Q33212). The proteins were then diluted into 8 mg / mL and 4 mg / mL stocks using the same cosolute solution. Due to the stipulation for each sample to have an identical buffer blank, the concentration of cosolute in the 8 mg / mL sample had to be the same as in the 4 mg / mL sample, meaning the molar ratio would be doubled in the 4 mg / mL sample. For each sample, a small aliquot of buffer was saved and stored at 4°C for use as a blank. Samples and buffer blanks were filtered using 0.22 pm syringe filters and loaded into an Axygen 96-well polypropylene PCR Microplate (Corning product# PCR-96-FS-C), which was then sealed with an AxyMat Sealing Mat (product# AM-96-PCR-RD) and wrapped in parafilm. Plates were shipped to Lawrence Berkeley National Labs in a styrofoam cooler filled with cold packs. All SAXS measurements were performed by the SIBYLS group at the Lawrence Berkeley National Laboratory HT-SAXS beamline (12.3.1) as described in Dyer KN et al., “High-throughput SAXS for the characterization of biomolecules in solution: apractical approach,” Methods Mol Biol, 2014, 1091 : 245-258; and Trame C et al., “SIBYLS — a SAXS and Protein Crystallography Beamline at the ALS,” AIP Conference Proceedings, American Institute of Physics, 2004, pp. 502-505. For technical restrictions, proteins were measured at 20: 1 and 200: 1 molar ratios of trehalose and sucrose instead of 10: 1 and 100: 1. The exception to this was CAHS D, which was tested with a wider range of molar ratios (1.6: 1, 16: 1, and 160: 1).

[0090] The radius of gyration calculated for CAHS D is approximately 5 angstroms greater than previously reported. This difference may be attributed to minor differences in the present approach. While Size Exclusion Chromatography (SEC)-coupled SAXS and a relatively dilute CAHS D sample was utilized in a previous study, the number of SAXS experiments in this study necessitated a higher-throughput approach that omitted the SEC step. While not wishing to be bound by any theory, it is therefore believed that the control samples contained a higher fraction of transient oligomeric species, which inflated the radius of gyration without significantly curving the Guinier region. Given that this fact was consistent between all of the CAHS D samples, the inter- environmental comparisons are still valid.1.L Small-Angle X-ray Scattering (SAXS) - Guinier and pMW Analysis

[0091] Notable aggregation, likely induced by exposure to X-rays, was present in some samples, especially in solutions that contained cosolutes. This was controlled for by excluding scattering data from samples that had already been exposed to large amounts of X-ray radiation and were thus statistically different from the initial readings. Despite some readings having been excluded, a Guinier analysis was able to be conducted for each protein-cosolute combination. Buffer subtractions and Guinier analysis were performed using BioXTAS RAW v. 2.1.4 as described in Nielsen SS et al., “BioXTAS RAW, a software program for high-throughput automated small-angle X-ray scattering data reduction and preliminary analysis,” J Appl Crystallogr, 2009, 42: 959-964; and Hopkins JB et al., “BioXTAS RAW: a free open-source program for small-angle X-ray scattering data reduction,” Foundations of Crystallography, 2018, 74: a219.

[0092] A qMaxRg of 1.1 was used to establish linear fits in the Guinier region as described in Navarro-Retamal C et al., “Molecular dynamics simulations and CD spectroscopy reveal hydration-induced unfolding of the intrinsically disordered LEA proteins CORI 5 A and COR15B from Arabidopsis thaliana,” Phys Chem Chem Phys,2016, 18: 25806-25816; and Rieloff E et al., “Determining Rgof IDPs from SAXS Data,” Methods Mol Biol, 2020, 2141 : 271-283. Samples with Guinier regions that could not be fit were excluded from the study. Molecular weight approximations were performed using the method described in Hajizadeh et al. 2018, which is programmed directly into BioXTAS RAW. See Hajizadeh NR et al., “Consensus Bayesian assessment of protein molecular mass from solution X-ray scattering data,” Sci Rep, 2018, 8: 7204. The 8 mg / mL samples tended to be far more aggregation-prone than the 4 mg / mL samples, so only the 4 mg / mL data is reported here. l.J. Photo-Induced Cross-Linking of Unmodified Proteins (PICUP)

[0093] PICUP crosslinking was performed as previously described in Rivera- Najera LY et al., “A group 6 late embryogenesis abundant protein from common bean is a disordered protein with extended helical structure and oligomer-forming properties,” J Biol Chem, 2014, 289: 31995-32009; and Liu G et al. “Involvement of C-Terminal Histidines in Soybean PM1 Protein Oligomerization and Cu2+ Binding,” Plant Cell Physiol, 2017, 58: 1018-1029. Briefly, lyophilized protein, Ru(II)bpy32+, and ammonium persulfate were resuspended in 20 mM Tris pH 7.5. Each reaction mixture constituted protein at the desired concentration with 1.25 mM Tris (2,2’- bipyridyl)dichlororuthenium(II)hexahydrate (Sigma, CAS No. 50525-27-4, and 2.5 mM ammonium persulfate (Sigma, CAS No. 7727-54-0) to a final volume of 10 pL. For mixtures, cosolutes and proteins were mixed at a 100: 1 molar ratio at 2X molar concentration. Photoreaction was triggered by flashing 72 W light through a 2.5 cm water filter for 10 seconds in a dark room. The reaction was immediately quenched by adding 10 pL of 2X Laemmli buffer containing 4 % SDS and 10 % P-mercaptoethanol. The reaction mixture was heated at 95 °C for 5 minutes. 8.5 pL of each sample was run in denaturing SDS-PAGE gels and stained with coomassie blue to visualize the oligomeric states. l.K. Differential Scanning Calorimetry (DSC) Measurements

[0094] Samples were prepared in Eppendorf tubes at the desired molar ratios with cosolutes. Protein mixtures were resuspended and incubated at 55°C for 5 minutes to ensure proper solubility. 25 pL of the sample was hermetically sealed into a previously massed pair of DSC aluminum hermetic pan and hermetic lid (Catalog 900793.901 and 901684.901, respectively, TA instruments). The sample mass was determined after the sample was sealed within the pan and lid. The sealed samples were then run on a TADSC2500 instrument. The DSC method for heating experiments is as follows: Samples were equilibrated at 20°C, heated to 60°C at a 5°C per minute ramp, and then cooled to 20°C at a 5°C per minute ramp. Samples were held for a 10 minute isothermal hold at 20°C and heated to 60°C at a 5°C per minute ramp. Trios software (TRIOS version #5.0.0.44608, TA Instruments) was used to analyze enthalpy for samples showing the melt curves. The changes in enthalpy for the mixtures were calculated relative to the protein. l.L. AlphaFoldl Structural Modelling

[0095] Protein structure prediction was performed using Google’s AlphaFold Collab notebook(https: / / colab. research. google.com / github / deepmind / alphafold / blob / main / not ebooks / AlphaFold. ipynb.). The setting “relax use gpu” was checked to increase the speed of individual simulations. The number of recycles was left at 3 as described in lumper I et al., “Highly accurate protein structure prediction with AlphaFold,” Nature, 2021, 596: 583-589. l.M. Transfer Free Energy (TFE) Calculations

[0096] TFE values of each cosolute for each amino acid were pulled from existing literature: Auton M et al., “Predicting the energetics of osmolyte-induced protein folding / unfolding,” Proc Natl Acad Set USA, 2005, 102: 15065-15068; Hong I et al. “Its preferential interactions with biopolymers account for diverse observed effects of trehalose,” Biophys J, 2015, 109: 144-153; and Auton M et al. “Additive transfer free energies of the peptide backbone unit that are independent of the model compound and the choice of concentration scale,” Biochemistry, 2004, 43: 1329-1342.

[0097] The inventor used experimentally derived TFE values for the transfer of a chemical group — amino acid side chains or backbone — into 1 M solutions of trehalose, sucrose, or glycine betaine as described in Auton M et al., “Application of the transfer model to understand how naturally occurring osmolytes affect protein stability,” Methods Enzymol, 2007, 428: 397-418; Auton M et al., Proc Natl Acad Sci USA, 2005, 102: 15065-15068; and Hong I et al. Biophys J, 2015, 109: 144-153.

[0098] The inventor then calculated the TFE for each conformation using the formula:

[0099] Here, AGtris the TFE of a protein conformation from water to 1 M cosolute solution, N is the chemical group, i is a numerical index for all instances of the chemical group, a is the surface area of the specific instance of the chemical group in square angstrom, and g is the experimental value of the transfer free energy for that chemical group per square angstrom of exposed surface area. By doing this for two conformations of a protein (e.g., for both the monomeric state G” and the dimeric state AG r), one can find the change in free energy of conformational change that can be attributed to the presence of an osmolyte.

[0100] The protein conformations utilized to perform the above equation were obtained from AlphaFold Multimer and AlphaFold 2, respectively (see AlphaFold method). Surface area for residues was calculated using SOURSOP, a python package for protein structure analysis as described in Lalmansingh JM et al., “SOURSOP: A Python package for the analysis of simulations of intrinsically disordered proteins,” bioRxiv, 2023, doi: 10.1101 / 2023.02.16.528879. To calculate thefor theLEA proteins and for BSA, the inventor compared an AlphaFold simulation of a monomeric protein with a theoretical conformation in which all residues are 100% exposed.

[0101] Here, AG"1utialrepresents the free energy of a completely denatured protein chain where the maximum theoretical accessibility is achieved (RASA = 1). Gltialrepresents the free energy of the protein’s “native” conformation (as determined by an AlphaFold simulation). AlphaFold simulations of the LEA proteins presented herein were broadly helical, and thus were used to represent the disorder-to- helix transition commonly observed in LEA proteins in all figures.

[0102] Two “end-state” protein conformations are used to calculate a AAGtrvalue, which is typically only possible for well-folded proteins. However, while not wishing to be bound by any theory, it is believed that the proteins used in this study constitute an exception. Several different groups have noted the propensity of CAHS proteins for oligomerization, and recent research has identified the dimer as a particularly stable CAHS D conformer. This notion is supported by the crosslinking data, in which the CAHS D dimer is especially prominent. For LEA proteins, the inventor instead optedfor an alpha-helical end-state. This was inspired by evidence that LEA protein undergoes a disorder-to-helix transition and supported by CD data.1.N. Data Analysis and Visualization

[0103] LDH protection was fitted into a sigmoidal curve by fitting a 5PL regression analysis using GraphPad Prism v9.5.1 from which the resulting PD50 values were derived. All other plots were plotted using R-Studio Version 2023.06.0+421. Annotations for statistical significance include: p>0.05: NS, p= 0.01-0.05: *, p= 0.001- 0.01 : **, p<0.001 : ***.2, Example IDPs Investigated

[0104] Amino acid sequences for selected IDPs are shown in Table 1 A. Amino acid sequences for selected LEA motif sequences are shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7. Amino acid sequences for selected full-length proteins are shown in SEQ ID NOs: 8, 9, 10, 11, 12, and 13.Table 1A

[0105] Nucleotide sequences for selected full-length proteins are shown in SEQ IDNOs: 8, 9, 10, 11, 12, and 13.

[0106] Table IB shows selected nucleotide sequences (SEQ ID NOs: 14, 15, 16, 17, 18, or 19) that encode the corresponding selected full-length proteins shown in Table 1A.Table IB

[0107] An amino acid sequence for a mutant construct (“FL-Proline) is shown in Table 1C. FL-Proline is an engineered variant (mutant construct) of the wild type CAHS D protein. It was made via the insertion of four prolines in the C-terminal domain of CAHS D. This disrupts beta-sheet structure in the C-terminal domain and abolishes the ability of the protein to form gels. Nonetheless, the data presented herein demonstrates that the protein remains protective and synergistic with disaccharides such as trehalose and sucrose.Table 1C3, Non-limiting Results3.A. Desiccation-related IDPs are enriched in organisms alongside specific cosolutes during drying

[0108] To test whether IDP sequences have evolved to be functionally-tuned by the composition of the intracellular environment during drying, the inventor selected six desiccation-related IDPs. These IDPs come from two LEA families (LEA l and LEA 4) as well as the CAHS family (Table ID). Table ID shows a summary of the disaccharides and proteins used in this study. Table ID also shows the organismal source of representative LEA 4, LEA l and CAHS proteins used in this study. In addition, the table displays endogenous cosolute reported in the literature to be coenriched alongside LEA and CAHS proteins during desiccation in the given organism (see enriched disaccharide during desiccation). The consensus sequence of 11-mer LEA 4 or 20-mer LEA l motifs as well as the length of the full-length proteins and predicted disorder using Metapredict were collected (disorder plots not shown). Shadedareas in the predicted disorder plot corresponded to the motif coordinates in the full- length LEA proteins. The reason many of these profiles contained large folded regions may be because the amphipathic LEA and CAHS proteins are predicted to form helices, which Metapredict infers and incorrectly highlights these regions as ‘folded’ when really they are disordered in isolation.Table ID

[0109] Four LEA 4 proteins, each from a different desiccation tolerant organism, were selected. These organisms included the plant Arabidopsis thaliana (AtLEA3-3), the nematode Aphelenchus avenae (AavLEAl), the tardigrade Hypsibius exemplar is (HeLEA68614), and the rotifer Adineta vaga (AvLEAlC) (Table ID). To assess whether synergy extended across LEA families, a LEA l protein from A. thaliana(AtLEA4-2) was selected (Table ID). CAHS D was selected from the tardigrade H. exemplaris (Table ID).3.B. LEA motifs and endogenous cosolutes during desiccation

[0110] To assess whether cosolutes induce functional changes in desiccation- related IDPs, the inventor began by testing peptides encoding LEA motifs derived from full-length LEA 4 and LEA l proteins (Table ID). Family 1 LEA (LEA l) proteins are characterized by a 20-mer repeating motif, whereas Family 4 LEA (LEA 4) proteins are characterized by the repetition of an 11-mer LEA 4 motif. These motifs are often found in multiple linear or nonlinear repeats across the length of a LEA protein. Interestingly, LEA 4 motifs have previously been suggested to be sufficient to confer desiccation protection to desiccation-sensitive proteins and membranes to a degree similar to full-length LEA proteins, both in vitro and in vivo. With this in mind, the inventor expected to observe synergy between LEA motif repeats and their paired cosolute(s).[OHl] The inventor generated 11-mer LEA 4 motif peptides (Atl 1, Aavl 1, Hel 1, and Avl 1) and a 20-mer LEA l peptide (At20) and measured the ability of these motifs to protect LDH — a desiccation-sensitive enzyme — during drying. LDH assay was used to assess the function of desiccation protectants to protect the activity of LDH which otherwise retains only approximately 2% of its pre-desiccation activity when dried and rehydrated.

[0112] The protective capacity for each LEA motif and cosolute was assessed across a range of concentrations with LDH. Most LEA 4 motifs displayed levels of protection so low that a 50% level of protection could not be reached even at concentrations exceeding 1 mM. Additionally, higher concentrations of the LEA 4 motifs tended to inactivate the enzyme when kept under control conditions (4°C, see methods), over a 16-hour incubation period during the assay.

[0113] The LEA l 20-mer motif At20, however, showed robust concentrationdependent protection of LDH, demonstrating that LEA 4 and LEA l motifs are functionally distinct. Concentration-dependent protection was also found for the cosolutes trehalose and sucrose.

[0114] Because LEA 4 motifs often exist in tandem repeats of 11-mers within a full-length LEA protein, the inventor examined whether the observed lack of protection was a result of their short length or repeat number. Here, a 2X (At22) and a 4X (At44)tandem repeats of the A. thaliana 11-mer LEA 4 motif (Atl 1) were synthesized. The results indicated minimal potency in preserving in vitro LDH function during drying regardless of motif length.

[0115] Despite the low protection displayed by the LEA 4 peptides, the LEA 4 peptides were used in cosolute synergy assays, as perhaps they may become functional when in solution with trehalose or sucrose. Concentrations of protectants were selected such that under instances of synergistic protection, the additive protection of cosolute:peptide mixtures would not exceed 100%. Table 3 shows protein concentrations used in synergy assays for LEA motifs (Atl l, Aavl l, Hel l, Avl l, At22, At44, and At20) in this study.Table 3

[0116] Synergy assays were then performed where sucrose or trehalose was combined with LEA motifs at molar ratios of 1 : 100, 1 : 10, 1 :1, 10: 1, and 100: 1 (cosolute: protein). The upper limit (10: 1 and 100: 1) of these ratios closely aligned with cosolute: protein ratios that produce synergistic protection between the tardigrade disordered protein CAHS D and trehalose. Here, the synergy is reported by showing the individual protective ability of the cosolute and IDP on its own, the sum of these protective values (hypothetical additive effect), and the actual measured protection produced by combination of the cosolute and peptide. The synergy was quantified using the following equation:% synergy = (% LDH protect! on)iDP+cosolute>(% LDH protect! on)mp - (% LDH protection)cosolute

[0117] In nearly all cases, synergy was not found for the 11-mer LEA 4 peptides with either sucrose or trehalose (FIG. 1 A and FIG. IB). In fact, in several cases, mixingLEA 4 peptides with sucrose or trehalose elicited antagonistic, rather than synergistic, effects (FIG. 1 A and FIG. IB). These results suggest that LEA 4 motifs do not robustly preserve LDH function, nor do they interact with cosolutes trehalose or sucrose in a functionally productive fashion. Similar to 11-mer motifs, the A. thaliana 22- and 44- mer peptides did not synergize with either trehalose or sucrose (FIG. 1 A and FIG. IB).

[0118] Unlike LEA_4 motifs, the 20-mer LEA_1 motif, At20, was both protective and synergized with both trehalose and sucrose (FIG. 1 A and FIG. IB).

[0119] Taken together, these experiments demonstrate a diversity in disordered protein / motif function, where LEA 4 motifs largely are not protective to the enzyme LDH during drying, nor are they synergistic with endogenous cosolutes. Conversely, the LEA l motif tested was highly protective and synergized with either sucrose or trehalose.3.C. Desiccation-related IDPs synergize with cosolutes

[0120] While LEA proteins are identified through homology in conserved LEA motif repeats, they also contain varying quantities of non-motif sequence (Table ID). Because it was found that that LEA 4 motifs generally provided relatively little protection and tended not to synergize with endogenous cosolutes in LDH assays, the inventor examined whether full-length LEA proteins might. Also included in the analysis was CAHS D.

[0121] The inventor began by testing the baseline protection of the proteins using the LDH assay. All full-length LEA l and LEA 4 proteins conferred protection for LDH activity up to the pre-desiccated value. Likewise, CAHS D provided concentration-dependent protection to LDH as previously observed. Bovine serum albumin (BSA) in these studies was utilized as a control.

[0122] Unlike the LEA motifs, most full-length proteins protected 50% LDH at concentrations less than 1 mM (and greater than 0 mM). Using data derived from the concentration range of LDH assays, the inventor selected a concentration that provided 15-45% protection for each protein to perform synergy experiments (Table 4). Table 4 shows protein concentrations used in synergy assays for full-length proteins — AtLEA3- 3, AavLEAl, HeLEA68614, AvLEAlC, AtLEA4-2, CAHS D, BSA— in this study.Table 4

[0123] It was found that nearly all full-length IDPs showed synergy with either sucrose or trehalose or both. Exceptions to this were AvLEAlC, which is derived from a rotifer that accumulates neither trehalose nor sucrose, and BSA, which comes from cows, which has no capacity for anhydrobiosis. Remarkably, in cases where LEA proteins displayed synergy, it was found that they were always more synergistic with endogenous compared to exogenous cosolutes.

[0124] Taken together, these experiments demonstrate that synergistic interactions between IDPs and cosolutes extended across multiple families of desiccation-protective IDPs found in a variety of organisms. It is also of note that, while many of the LEA motifs tested at the beginning of this study did not display synergy with trehalose or sucrose, the corresponding full-length proteins did. Likewise, At20 showed synergy with both trehalose and sucrose, while full-length AtLEA4-2 was only synergistic with sucrose. Thus, not only do these experiments show that full-length LEA proteins synergized with their endogenous cosolute(s), but they demonstrate that this synergy, at least in part, is driven by sequence features beyond conserved motifs.3.D. Effect of trehalose and sucrose on local ensemble changes to desiccation- related IDPs in solution and in the dry state

[0125] The inventor next examined what mechanism(s) drive the functional synergy found between desiccation-protective IDPs and cosolutes. The inventor reasoned that functional synergy might be driven by cosolute-induced changes to the IDP ensemble. To test this, the secondary structure contained in the ensemble of LEA proteins and CAHS D was first examined using circular dichroism (CD) spectroscopy.

[0126] Each full-length protein was first assessed using CD in an aqueous state by itself. All full-length LEA proteins displayed a single minimum at -200 nm indicating that LEA proteins are disordered in the aqueous state. CAHS D also displayed a minimum at -200 nm and a slight minimum around -220 nm, indicating that whiledisordered, it also had some propensity for helical structure in solution. This is in contrast to BSA, which showed a high propensity for helical structure as denoted by the double minima at 222 and 210 nm. To determine whether the addition of cosolutes induces secondary structural change in the aqueous state, CD spectra of cosolute: protein mixtures at 100: 1 molar ratios were obtained. Surprisingly, trehalose and sucrose did not induce any significant structural changes to any of the full-length IDPs tested here.

[0127] LEA proteins gain helical conformation upon drying or in response to low water availability. Drying-induced helicity has been postulated to drive their protective function. The inventor reasoned that while the cosolutes did not induce detectable changes to LEA / CAHS secondary structure in solution, cosolutes could induce structural changes in proteins in a dry state. To test this, the proteins were examined using CD in a desiccated state. The results showed a significant structural change for all LEA proteins and CAHS D in the dry state, indicated by a shift from disordered spectra with a minimum at -200 nm to a helical structure with two minima at -222 and -210 nm. This is in contrast to BSA which started out helical and showed little change in the spectrum. These changes manifested for pure proteins without any addition of synergistic cosolutes. To quantify the influence of drying on secondary structure, the changes in the ratio of CD signal at 222 and 210 nm were examined. This ratiometric value reports on secondary structure in a concentration-independent way. Using this metric, all LEAs and CAHS D displayed a significant change in structure going from the aqueous to dehydrated state, whereas BSA remains the same.

[0128] Next, combinations of the proteins with trehalose or sucrose in a desiccated state were examined. As with the aqueous samples, the addition of trehalose or sucrose did not induce significant changes in the secondary structure of LEA and CAHS D proteins in the dry state. To assess whether there is a link between the minimal structural changes observed and functional synergy in LDH assays, the change in the ratio of signal at 210 and 222 nm in desiccated and aqueous states was compared to the synergy observed for that same mixture. Synergistic protection observed in the LDH assays did not correlate with secondary structural changes with the addition of trehalose (p=0.9863 for aqueous, p=0.1113 for desiccated) or sucrose (p= 0.6673 for aqueous, p=0.9863 for desiccated).

[0129] Taken together, these results indicate that, while LEA proteins and CAHS D undergo a structural transition during desiccation, this phenomenon does not require, nor is it affected by, the presence of trehalose and sucrose. Furthermore, synergistic protection observed in the LDH assays may not be mediated by local ensemble changes in these IDPs.3.E. Effect of trehalose and sucrose on global ensemble dimensions for desiccation- related IDPs, and oligomerization of CAHS D

[0130] Next examined was whether the synergistic interactions found between the IDPs and cosolutes could instead be explained by a change in global dimensions, such as expansion or compaction of the protein. Small angle X-ray scattering (SAXS) was used to measure global dimensions. SAXS allows a model-free estimation of the radius of gyration (Rg) of an IDP as well as a prediction of the molecular weight. Each protein was measured with no cosolute and with different molar ratios of trehalose and sucrose. The inventor reasoned that a cosolute-dependent change in the radius of gyration (Rg) could indicate changes in tertiary or quaternary structure, which may correlate with increased function or synergy. For SAXS experiments, cosolutes were used at concentrations between 20 mM and 50 mM due to technical restrictions on how much protein can accurately be assayed and a desire to maintain molar ratios used in other experiments.

[0131] The inventor began by testing the Rgof BSA in different solution conditions. As a well-folded protein, it was expected that BSA would be relatively insensitive to changes in the solution environment. The Rgvalues obtained via this approach matched existing literature. Additionally, adding cosolutes did not modulate Rg. While the molecular weight approximations trended higher than expected for monomeric BSA, this may be due to the propensity of BSA to form small populations of low-level oligomers. The Rgof the LEA proteins studied in various solution conditions were then measured. It was found that, regardless of the solution environment, all of the LEA proteins had an Rgthat falls within error of readings in other solution environments. At the concentrations used here, cosolutes did not induce significant changes in the global dimensions of LEA proteins. While the predicted molecular weight (pMW) from SAXS for these proteins were somewhat variable, no consistent trend was found between the presence of cosolutes and change in pMW for any LEA protein.

[0132] Finally, CAHS D was tested in similar solution conditions. An Rgvalue for CAHS D that lies between the values reported by other groups was obtained (see methods). While this Rgvalue was consistent in 1.6: 1 disaccharide solutions, 16: 1 disaccharide solutions, and 160: 1 sucrose solution, the inventor found that the 160: 1 trehalose solution had a Guinier region that was sharply curved upwards, even upon the protein’s first exposure to the X-ray source (FIG. 2). This result is consistent with the presence of large oligomerized species. A Bayesian approximation of the molecular weight of these samples supported this result (FIG. 2). Most of the CAHS D samples showed a pMW that was only slightly elevated from the known value for the monomeric protein. In contrast, the 160: 1 sucrose sample was found to have a pMW about 300% higher than other samples, and the 160: 1 trehalose sample had a pMW about 1000% higher (FIG. 2). Overall, the results indicated that cosolutes increase the global dimensions of CAHS D, but not LEA or BSA.

[0133] While SAXS showed little change in Rgor pMW for LEA proteins, there was evidence that not only CAHS D but also some LEA proteins tend to oligomerize. However, LEA oligomerization appeared to be weak and transient, requiring extreme crowding and / or sensitive methods to detect. Therefore, a more sensitive method was used to assess LEA oligomers and how they might be affected by cosolutes.

[0134] Photo-induced crosslinking of unmodified proteins (PICUP) was used to characterize oligomerization of LEA proteins in a more sensitive fashion. PICUP is a zero-length crosslinking method that uses a light activatable crosslinking system and is known to capture transient oligomeric species. All of the LEA proteins showed a propensity to form oligomers, even at low concentrations. However, the presence of trehalose or sucrose did not elicit changes in oligomeric populations. These results confirmed that LEA proteins are able to form transient oligomers. These results also demonstrated that, at levels where sucrose and trehalose are synergistic with these proteins, oligomerization was unaffected.

[0135] Taken together, these results show a divergence in the behavior of LEA and CAHS proteins in the presence of synergistic cosolutes. For CAHS D, the SAXS data suggests a relationship between the presence of cosolutes and increased oligomerization. However, this dataset showed no evidence of a cosolute inducible increase in molecular weight or Rgfor LEA proteins. This was further supported by PICUP, which despite detecting LEA oligomers did not show that they were enhancedby cosolutes. Thus, the data suggests that CAHS D oligomerization may be promoted by the presence of synergistic cosolutes while LEA oligomerization is not.3.F. Synergistic cosolutes promote gelation of CAHS D

[0136] The inventor examined whether the cosolute induced oligomerization of CAHS proteins in the presence of trehalose as seen in the SAXS experiments could be attributed to the propensity of CAHS D to form gels. To test this, differential scanning calorimetry (DSC) was performed on CAHS D to observe the presence or absence of a gel melt.

[0137] To begin, the inventor tested CAHS D at 6 mg / mL (0.235 mM). At 0.235 mM, it was found that CAHS D does not undergo a characteristic gel melt indicating a lack of gelation (FIG. 3 A). Hence 0.235 mM CAHS D is a non-gelling concentration. Addition of trehalose and sucrose at increasing molar ratios (1 : 1, 10: 1, 100: 1, and 500: 1 (disaccharide:CAHS D)) showed thermal features characteristic of endothermic phase transitions (e.g., a gel melting), indicating that the presence of these cosolutes induced gelation (FIG. 3A).

[0138] Measuring the area under these melt curves allows calculation of the enthalpy of melting. Change in enthalpy measurements for the cosolute :protein mixtures relative to the protein provide a quantification of how gelation is affected by different amounts of cosolutes. It was found that trehalose induced significant gelation at a 100:1 ratio (trehalose:IDP), while 500: 1 of sucrose:IDP was required to induce a significant gel melt under the conditions tested (FIG. 3B). This result was consistent with trehalose producing larger oligomeric species in the SAXS experiments (FIG. 2), indicating that trehalose had a larger influence than sucrose on the gelation of CAHS D (FIG. 3B).

[0139] Further, to test whether synergistic cosolutes enhance gelation, the inventor tested CAHS D at 12 mg / mL (0.47 mM), a concentration above CAHS D’s gelation threshold. Addition of trehalose or sucrose at increasing molar concentrations was found to promote the formation of stronger gels evident by the enthalpy of melting measurements. These experiments demonstrate that, not only do trehalose and sucrose induce sub-gelling concentrations of CAHS D to form gels, but they also enhance the strength of gels formed by higher concentrations of the protein.

[0140] Unlike CAHS proteins, gelation of LEA proteins has not been commonly observed or reported. The exception to this is AfrLEA6 (a LEA protein from the brineshrimp A. franciscanci) that appears to undergo phase separation, forming a hydrogellike matrix upon desiccation. To determine whether LEA proteins gel, DSC experiments were performed on the LEA proteins and cosolutes. Here, similar molar concentrations of LEA proteins on their own and in mixtures with cosolutes at equivalent ratios (1 : 1, 10: 1, 100: 1, and 500: 1 (disaccharide:LEA)) were analyzed. It was found that none of the LEA proteins by themselves or in mixtures with trehalose or sucrose showed evidence of gelation (data not shown). Likewise, BSA also failed to form a gel (data not shown).

[0141] Overall, the data indicated that cosolutes promote gelation of CAHS D at non-gelling concentrations, but that cosolutes may not promote gelation of LEA or B SA proteins at non-gelling concentrations. Taken together, these results demonstrate that trehalose and sucrose affected the oligomerization and phase state of different IDPs in distinct ways. The observation that trehalose induces more synergy as well as more gelation of CAHS D relative to sucrose may indicate that gelation and synergistic protection of LDH may be linked.3.G. Direct cosolute:IDP interactions drive synergy for CAHS D

[0142] To explain the possible relationship between synergy and oligomerization- driven gelation of CAHS D, the inventor quantified the interactions between each IDP and its cosolute environment using transfer free energies (TFEs). The TFE is a measure of the change in free energy undergone by a biological material of interest (e.g., a macromolecule) when transferring from water to a concentrated solution of some osmolyte (typically 1 M). Using transfer free energy values derived from literature, the inventor calculated the effect of trehalose and sucrose on the ability of CAHS D to dimerize: AAG^D. This is calculated by finding the free energy of CAHS D’s monomeric state upon transfer to an osmolyte solution (AG^.), doing the same for the dimeric state (AG^A), and then taking the difference. A strong negative value for AAG^Dindicates that the presence of the cosolute pushes the population towards dimers. A positive value indicates that the addition of the cosolute pushes the population towards monomers.

[0143] In order to perform these calculations, the inventor utilized AlphaFol d2 and AlphaFold Multimer to determine plausible conformations for both CAHS D’s monomeric and dimeric state. These structures have a helical linker region, which is consistent with the CD measurements and is consistent with previous reports for thisprotein. The inventor reasoned that dimerization is indicative of gelation since CAHS D dimers are especially prevalent in crosslinking data, and previous research suggests that CAHS D dimer formation may be a necessary step toward gelation. TFEs were then calculated based on the solvent accessible surface area of different chemical groups in monomeric vs. dimeric state (see methods above).

[0144] The calculations revealed that trehalose had a negative AAG^Dwith CAHS D, meaning the dimeric state is favored in the presence of this cosolute. Sucrose’s A G^Dwas close to 0, neither stabilizing nor destabilizing the dimer. In addition to these cosolutes, the effect of a cosolute with a positive AAG^Dwas explored. Glycine betaine, a common stabilizing cosolute, but without known roles in tardigrade desiccation tolerance, displayed a positive AAG^D. Together these cosolutes span a range of A A Gvalues that are expected to increase the dimer population (trehalose), have a minimal impact on dimerization (sucrose), or increase the monomer population (glycine betaine) of CAHS D. While data for trehalose and sucrose were in line with this analysis, the inventor next sought to determine empirically the impact of glycine betaine on CAHS D gelation.

[0145] To test the effects of glycine betaine on CAHS D dimerization predicted by these TFE calculations, the DSC and SAXS experiments were repeated in the presence of glycine betaine. Unlike trehalose and sucrose, below the protein’s gelation threshold no increase in enthalpy of melting was found upon the addition of glycine betaine (FIG. 4A). To probe whether glycine betaine inhibits CAHS D oligomerization, the inventor conducted additional DSC experiments above the protein’s gelation threshold. While trehalose and sucrose enhanced gelation of CAHS D, a decrease in enthalpy of melting was found when glycine betaine was present at the 500:1 molar ratio (glycine betaine:CAHS D), signifying inhibition of CAHS D gelation (FIG. 4B).

[0146] SAXS on CAHS D with 1000: 1 molar ratios of each cosolute was then performed. The intent of using such a high molar ratio was to mimic the high concentration of cosolutes that CAHS D would experience during desiccation. A nongelling concentration of CAHS D in 1000: 1 glycine betaine yielded a scattering profile similar to the protein with no cosolute indicating a lack of gelation. Meanwhile, 1000: 1 sucrose and trehalose yielded scattering profiles consistent with gelation, similar to those previously reported. This is made especially evident by a peak at q=0.06 A which reports on the width of CAHS D’s gel fibers and matches previously reportedscattering profiles for gelled CAHS D. Consistent with the inventor’s hypothesis that the more negative AAG^Dfor trehalose will increase dimerization and subsequent gelation, an increased curvature in the Guinier region was found, indicating increased fibrillization.

[0147] Finally, the impact of glycine betaine on CAHS D’ s protective capacity was tested. If induction of self-assembly of CAHS D is a mechanism underlying trehalose / sucrose induced synergy, then one would expect that glycine betaine’s inhibition of gelation would result in no synergy, or even have an antagonistic effect. A significant antagonistic relationship between glycine betaine and CAHS D on LDH protection was found (FIG. 4C). Furthermore, Pearson correlation between the AG^Dof a cosolute (scaled by concentration) and its ability to induce synergy in CAHS D was statistically significant (FIG. 4D). This correlation however did not hold for LEA proteins or for B SA.

[0148] Taken together, these results suggest that the driving force for gelation in CAHS D and its ability to synergize with a given cosolute may be rooted in the direct interaction between CAHS D and the prevalent cosolute. This observation ruled out the possibility for the combined interactions of the CAHS D: cosolute on the client protein (LDH). However, trying to apply this model to the LEAs tested in this work failed to yield meaningful correlates. Thus, it is proposed that direct interactions between cosolute and LEA proteins cannot explain the synergy observed with cosolutes. Overall, the study described herein demonstrates that, while synergy between desiccation- related IDPs and cosolutes appears to be a widespread and conserved behavior, the mechanisms underlying this synergy vary between IDP families.

[0149] FIGS. 5A-5C show non-limiting synergy plots for various cosolutes with FL-proline as a function of molar ratio (cosolute :FL-proline). arious molar ratios of cosolute:FL-proline were tested: 1 : 1, 10: 1, 100: 1 and 500:1. FIG. 5A shows data for trehalose :FL-proline, FIG. 5B shows data for sucrose:FL-proline, and FIG. 5C shows data for betaine:FL-proline. While the onset of synergy is slightly delayed (observing synergy at 1 : 100 rather than 1 : 10 with the wild type protein), significant and robust synergy with trehalose and sucrose is observed. As with the other proteins, betaine is not synergistic, but rather antagonistic.4, Non-limiting Discussion

[0150] In this study, the interplay between IDP sequence, solution environment, ensemble, and function was examined. To do this, the inventor has taken advantage of the dramatic changes to the cosolute content of anhydrobiotic organisms brought on by desiccation, and compared the effects of these cosolutes between three families of desiccation-related IDPs (LEA 4, LEA l, and CAHS proteins). The data presented herein demonstrates that endogenous cosolutes enriched during desiccation enhance the protective capacity of CAHS D, full-length LEA 4, and LEA l proteins in an in vitro enzyme assay. Surprisingly, the functional changes were not accompanied by any detectable structural changes to the monomeric ensemble of these IDPs. However, synergistic cosolutes did induce oligomerization and gelation of CAHS D. Finally, the data shows that in the case of CAHS D, but not LEAs, oligomerization, gelation, and protective synergy can be traced to direct interactions between cosolute and protective protein. The results presented herein suggest that, while functional synergy between the solution environment spans multiple IDP families, different mechanisms can underlie synergistic interactions for different proteins.

[0151] Functional synergy for the full-length proteins from different organisms mirrored the endogenous cosolute environment in that organism. In all cases, an IDP protected LDH activity more with its endogenous cosolute compared to an exogenous cosolute. These differences in synergy appear to extend across even subtle variations in cosolute use. For example, nematodes and tardi grades both accumulate trehalose during desiccation, but tardigrades accumulate orders of magnitude less. Consistent with this, both tardigrade proteins used in this study synergized with trehalose at an order of magnitude lower concentration than what was required to elicit synergy with the nematode LEA protein.

[0152] Overall, while the present study found that synergy with endogenous cosolutes is observed across two families of LEA proteins as well as CAHS proteins, a major difference in structural changes induced in these protein families was observed. While in CAHS D synergy could be traced back to the interaction between the protective protein and the cosolute, no underlying mechanism was detected for LEAs. What then could be driving the synergy observed for these proteins?

[0153] One possibility is that the protein being protected is not considered in most of the assays. The studies of molecular mechanisms for synergy did not consider the underlying effect of both protectant protein and cosolute on LDH directly. It is possiblethat the presence of both endogenous cosolute and protein create a solvation environment that becomes highly protective for e.g. rehydration. In line with this, recent studies have highlighted the ability of LEA proteins to stabilize sugar glasses in a dry state. Glass formation can preserve labile biomolecules during desiccation, contributing to survival. Different glasses vary significantly in their protective capacity, and studies have attempted to find structural properties that explain this difference. Because trehalose and sucrose both form glasses when dried, it is possible that the LEA proteins are inducing a change in the glass’s structural properties that leads to synergy.

[0154] Another possibility is a difference in the nature of TFE-induced oligomerization. While repulsive cosolutes drive homotypic interactions between CAHS D monomers, the same thermodynamic force can promote heterotypic interactions between LEAs and other proteins. For example, trehalose may stabilize electrostatic interactions between LEA proteins and LDH during the in vitro synergy assays. If the protective capacity of LEA proteins is dependent on direct interactions between the protectant and the client protein, then this is a plausible explanation for synergy. However, it is currently unknown whether or not this is the case.

[0155] Another major question posed by this research is why sucrose was able to elicit synergy in CAHS D. The computational approach predicted that sucrose should be agnostic to CAHS D’s ability to form dimers, and yet it was clearly seen that, in vitro, sucrose is a moderately potent driver of gelation. Several factors may explain this effect. While sucrose does not drive dimerization through direct ‘soft’ repulsion, it may still do so by acting as a crowder eliciting an excluded volume effect. Another possible manifestation of an excluded volume effect is the slight increase in the melting peak that is observed with all cosolutes used in this study at high molar ratios. Alternatively, given that CAHS D polymerizes beyond the dimeric state to form a gel, sucrose may stabilize a higher-level oligomer that was not captured in the AAG^Danalysis.

[0156] IDPs play regulatory functions during development and disease progression that often occurs together with changes to the chemical composition of the intracellular environment. For example, there may be links between type II diabetes and Alzheimer’ s disease, and it has been shown that the intrinsically disordered neurodegenerative peptide Ap42 undergoes pathological oligomerization in the presence of glucose whose levels mirror those found in diabetic patients. The study presented herein showcases how different cosolute environments can have a direct effect on the function of IDPs.By understanding the rules governing desiccation related IDP-cosolute interactions, the influence of changing chemical environments on a host of other IDPs may be better understood.Embodiments Listing

[0157] The present disclosure provides, among others, the following embodiments, each of which can be considered as optionally including any alternate embodiments:

[0158] Embodiment 1. A method, comprising: introducing an exogenous cosolute with an intrinsically disordered protein (IDP) to induce aggregation or oligomerization of the IDP, the IDP comprising an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof.

[0159] Embodiment 2. The method of Embodiment 1, wherein the IDP comprises an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 13 or 20.

[0160] Embodiment 3. The method of any one of Embodiments 1-2, wherein the exogenous cosolute comprises a disaccharide.

[0161] Embodiment 4. The method of Embodiment 3, wherein the disaccharide comprises trehalose, sucrose, maltose, lactose, or combinations thereof.

[0162] Embodiment 5. The method of any one of Embodiments 3-4, wherein the disaccharide comprises trehalose, sucrose, or combinations thereof.

[0163] Embodiment 6. The method of any one of Embodiments 3-5, wherein the disaccharide is introduced in an amount sufficient to induce gelation of the IDP.

[0164] Embodiment 7. The method of any one of Embodiments 3-6, wherein a molar ratio of the disaccharide to the IDP is greater than 10: 1.

[0165] Embodiment 8. The method of any one of Embodiments 3-7, wherein a molar ratio of the disaccharide to the IDP is in a range from about 50: 1 to about 1,000: 1, such as from about 100:1 to about 500: 1.

[0166] Embodiment 9. A method of inducing aggregation or oligomerization of a protein, the method comprising: introducing an exogenous disaccharide with a monomeric intrinsically disordered protein (IDP) to form a composition comprising the exogenous disaccharide and an aggregated form or oligomerized form of the IDP, the monomeric form of the IDP comprising an amino acid sequence having at least 90% identity to one or more ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof, a molar ratio of the exogenous disaccharide to the IDP is in a range from about 10:1 to about 1,000: 1, such as from about 50: 1 to about 1,000: 1, such as from about 100: 1 to about 500:1.

[0167] Embodiment 10. The method of Embodiment 9, wherein: the monomeric form of the IDP comprises an amino acid sequence having at least 90% identity to one or more of SEQ ID NOs: 13 or 20; and / or the exogenous disaccharide comprises trehalose, sucrose, or a combination thereof.

[0168] Embodiment 11. A composition, comprising: an oligomeric form of an IDP, the IDP comprising an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof; and an exogenous cosolute.

[0169] Embodiment 12. The composition of Embodiment 11, wherein the oligomeric form of the IDP comprises a dimeric form of the IDP, a trimeric form of the IDP, a tetrameric form of the IDP, or combinations thereof.

[0170] Embodiment 13. The composition of any one of Embodiments 11-12, wherein the IDP comprises an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 13 or 20.

[0171] Embodiment 14. The composition of any one of Embodiments 11-13, wherein the exogenous cosolute comprises a disaccharide.

[0172] Embodiment 15. The composition of Embodiment 14, wherein the disaccharide comprises trehalose, sucrose, maltose, lactose, or combinations thereof.

[0173] Embodiment 16. The composition of any one of Embodiments 14-15, wherein the disaccharide comprises trehalose, sucrose, or combinations thereof.

[0174] Embodiment 17. The composition of any one of Embodiments 14-16, wherein a molar ratio of the disaccharide to the IDP is greater than 10: 1.

[0175] Embodiment 18. The composition of any one of Embodiments 14-17, wherein a molar ratio of the disaccharide to the IDP is in a range from about 50: 1 to about 1,000: 1, such as from about 100: 1 to about 500: 1.

[0176] Embodiment 19. The composition of any one of Embodiments 11-18, further comprising 15 wt% or less of water based on a total wt% of the composition(such as from about 5 wt% to about 12 wt%), the total wt% of the composition is 100 wt%.

[0177] Embodiment 20. The composition of any one of Embodiments 11-19, further comprising a biological material of interest, the biological material of interest different from the IDP.

[0178] Embodiment 21. A method, comprising: introducing an exogenous cosolute with an intrinsically disordered protein to induce aggregation or oligomerization of the intrinsically disordered protein.

[0179] Embodiment 22. The method of Embodiment 21, wherein the intrinsically disordered protein comprises an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof.

[0180] Embodiment 23. The method of any one of Embodiments 21-22, wherein the exogenous cosolute comprises a disaccharide.

[0181] Embodiment 24. The method of any one of Embodiments 21-23, wherein the disaccharide comprises trehalose, sucrose, maltose, lactose, or combinations thereof.

[0182] Embodiment 25. An intrinsically disordered protein, comprising: an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof.

[0183] Embodiment 26. A nucleic acid comprising a nucleotide sequence encoding the intrinsically disordered protein of Embodiment 25.

[0184] Embodiment 27. The nucleic acid of Embodiment 26, wherein the nucleotide sequence has at least 80% identity to one or more of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, or a complement thereof.

[0185] Embodiment 28. The intrinsically disordered protein of any one of Embodiments 25-27 for stabilizing a biological material of interest.

[0186] Embodiment 29. A composition, comprising: an oligomeric form of an intrinsically disordered protein; and an exogenous cosolute.

[0187] Embodiment 30. The composition of Embodiment 29, wherein the intrinsically disordered protein comprises an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof.

[0188] Embodiment 31. The composition of any one of Embodiments 29-30, wherein the intrinsically disordered protein is encoded by a nucleotide sequence having at least 80% identity to one or more of SEQ ID NOs: 14, 15, 16, 17, 18, 19, or a complement thereof.

[0189] Embodiment 32. The composition of any one of Embodiments 29-31, wherein the oligomeric form of the intrinsically disordered protein comprises a dimeric form, a trimeric form, a tetrameric form, or combinations thereof.

[0190] Embodiment 33. The composition of any one of Embodiments 29-32, wherein the exogenous cosolute comprises a disaccharide.

[0191] Embodiment 34. The composition of Embodiment 33, wherein the disaccharide comprises trehalose, sucrose, maltose, lactose, or combinations thereof.

[0192] Embodiment 35. The composition of any one of Embodiments 29-34, further comprising: a biological material of interest, the biological material of interest different from the intrinsically disordered protein.

[0193] Embodiment 36. The composition of any one of Embodiments 29-35, further comprising 15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%.

[0194] Embodiment 37. A method for stabilizing a biological material of interest, the method comprising: introducing a biological material of interest with the composition of any one of Embodiments 29-36, wherein the composition is characterized as stabilizing the biological material of interest in a dry state at a temperature of ambient temperature or higher; and at least partially drying or desiccating the resultant mixture comprising the biological material of interest and the composition.

[0195] As is apparent from the foregoing general description and the specific aspects, while forms of the aspects have been illustrated and described, various modifications can be made without departing from the spirit and scope of the present disclosure. Accordingly, it is not intended that the present disclosure be limited thereby. Likewise, the term “comprising” is considered synonymous with the term “including.” Likewise whenever a composition, process operation, process operations, an element or a group of elements is preceded with the transitional phrase “comprising,” it is understood that we also contemplate the same composition or group of elements withtransitional phrases “consisting essentially of,” “consisting of,” “selected from the group of consisting of,” or “Is” preceding the recitation of the composition, process operation, process operations, element, or elements and vice versa, such as the terms “comprising,” “consisting essentially of,” “consisting of’ also include the product of the combinations of elements listed after the term.

[0196] For purposes of this present disclosure, and unless otherwise specified, all numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and consider experimental error and variations that would be expected by a person having ordinary skill in the art. For the sake of brevity, only certain ranges are explicitly disclosed herein. However, ranges from any lower limit may be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit may be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit may be combined with any other upper limit to recite a range not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the subranges 1 to 4, 1.5 to 4.5, 1 to 2, among other subranges. As another example, the recitation of the numerical ranges 1 to 5, such as 2 to 4, includes the subranges 1 to 4 and 2 to 5, among other subranges. Additionally, within a range includes every point or individual value between its end points even though not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the numbers 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, among other numbers. Thus, every point or individual value may serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited.

[0197] As used herein, the indefinite article “a” or “an” shall mean “at least one” unless specified to the contrary or the context clearly indicates otherwise. For example, aspects comprising “a cosolute” includes aspects comprising one, two, or more cosolutes, unless specified to the contrary or the context clearly indicates only one cosolute is included.

[0198] While the foregoing is directed to aspects of the present disclosure, other and further aspects of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

ClaimsWhat is claimed is:

1. A method, comprising: introducing an exogenous cosolute with an intrinsically disordered protein (IDP) to induce aggregation or oligomerization of the IDP, the IDP comprising an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof.

2. The method of claim 1, wherein the IDP comprises an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 13 or 20.

3. The method of claim 1, wherein the exogenous cosolute comprises a di saccharide.

4. The method of claim 3, wherein the disaccharide comprises trehalose, sucrose, maltose, lactose, or combinations thereof.

5. The method of claim 3, wherein the disaccharide comprises trehalose, sucrose, or combinations thereof.

6. The method of claim 3, wherein the disaccharide is introduced in an amount sufficient to induce gelation of the IDP.

7. The method of claim 3, wherein a molar ratio of the disaccharide to the IDP is greater than 10: 1.

8. The method of claim 3, wherein a molar ratio of the disaccharide to the IDP is in a range from about 50: 1 to about 1,000: 1.

9. A method of inducing aggregation or oligomerization of a protein, the method comprising: introducing an exogenous disaccharide with a monomeric intrinsically disordered protein (IDP) to form a composition comprising the exogenous disaccharideand an aggregated form or oligomerized form of the IDP, the monomeric form of the IDP comprising an amino acid sequence having at least 90% identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, or a complement thereof, a molar ratio of the exogenous disaccharide to the IDP is from about 10: 1 to aboutI,000:1.

10. The method of claim 9, wherein: the monomeric form of the IDP comprises an amino acid sequence having at least 90% identity to one or more of SEQ ID NOs: 13 or 20; and the exogenous disaccharide comprises trehalose, sucrose, or a combination thereof.I I. A composition, comprising: an oligomeric form of an IDP, the IDP comprising an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11. 12, 13, 20, or a complement thereof; and an exogenous cosolute.

12. The composition of claim 11, wherein the oligomeric form of the IDP comprises a dimeric form of the IDP, a trimeric form of the IDP, a tetrameric form of the IDP, or combinations thereof.

13. The composition of claim 11, wherein the IDP comprises an amino acid sequence having at least 80% identity to one or more of SEQ ID NOs: 13 or 20.

14. The composition of claim 11, wherein the exogenous cosolute comprises a di saccharide.

15. The composition of claim 14, wherein the disaccharide comprises trehalose, sucrose, maltose, lactose, or combinations thereof.

16. The composition of claim 14, wherein the disaccharide comprises trehalose, sucrose, or combinations thereof.

17. The composition of claim 14, wherein a molar ratio of the disaccharide to the IDP is greater than 10: 1.

18. The composition of claim 14, wherein a molar ratio of the disaccharide to the IDP is from about 50: 1 to about 1,000: 1.

19. The composition of claim 11, further comprising 15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition is 100 wt%.

20. The composition of claim 11, further comprising a biological material of interest, the biological material of interest different from the IDP.

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