Sustainable protein waste regeneration via entropy-driven denaturation

Abstract

The present invention provides methods for regenerating keratin from keratin-rich sources and uses thereof for fabricating, e.g., shape-memory polymeric fibers, yams, threads, fabrics, structures and objects. In one embodiment, it includes a method for preparing regenerated keratin comprising: (a) providing a keratin-rich source; (b) contacting the keratin-rich source with an aqueous solution comprising a denaturing agent and a reducing agent; (c) heating the mixture; (d) subjecting the extracted mixture to hot filtration; (e) cooling the keratin solution; (f) centrifuging the cooled keratin solution to obtain a subnatant comprising a denatured keratin gel and a supernatant comprising a solution of the denaturing agent; (g) reconstituting the subnatant comprising the denatured keratin gel in water to yield regenerated keratin; and (h) recycling the supernatant comprising a solution of the denaturing agent for use in steps (a) to (e), thereby preparing the regenerated keratin.

Description

DOCKET NO.117823-37020 TITLE SUSTAINABLE PROTEIN WASTE REGENERATION VIA ENTROPY-DRIVEN DENATURATION RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No.63 / 657,154, filed on June 7, 2024, the entire contents of which are incorporated herein by reference. STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government Support under 2011754 and 2025158 awarded by National Science Foundation (NSF), and under GM139571 and EY030444 awarded by National Institutes of Health (NIH). The Government has certain rights in this invention. BACKGROUND OF THE INVENTION

[0003] The intricate interactions between proteins, water and solutes determine not only protein structures, but also protein folding and functions. The underlying mechanisms have been a long-standing interest, but still unsolved questions, to a broad community ranging from physical chemistry and biophysics to molecular biology since the initial observation of the ‘Hofmeister Series’ in the 18thcentury (Hofmeister, F. Zur Lehre von der Wirkung der Salze. Archiv f. experiment. Pathol. u. Pharmakol 24, 247–260 (1888); Kunz, W., Henle, J. & Ninham, B. W. ‘Zur Lehre von der Wirkung der Salze’ (about the science of the effect of salts): Franz Hofmeister’s historical papers. Current Opinion in Colloid & Interface Science 9, 19–37 (2004)).

[0004] Significant progresses have been made in understanding the direct interactions between proteins or water and solutes, the origins of hydrophobicity and its effects on protein folding. However, there is still a lack of the unified framework of triadic relationship among proteins, water, and solutes, the critical parts of which are to decouple the direct and indirect solute effects (Xie, W. J. & Gao, Y. Q. A Simple Theory for the Hofmeister Series. J. Phys. Chem. Lett.4, 4247–4252 (2013)), and to experimentally and theoretically confirm the indirect solute effects, manifested as water hydrogen-bond network changes and quantified as water entropy, on protein structure integrity and folding processes. 1 ME1\53235995.v1DOCKET NO.117823-37020

[0005] Moreover, the unclear mechanisms have also immensely hindered the control, mass-production, and utilization of sustainable protein-based materials from renewable resources (Ashworth, C. Plastics from proteins. Nat Rev Chem 6, 165– 165 (2022); Rosenboom, J.-G., Langer, R. & Traverso, G. Bioplastics for a circular economy. Nat Rev Mater 7, 117–137 (2022). For instance, due to the limited understanding of protein-water-solute interactions, current protein extraction processes heavily rely on non-recyclable organic chemicals (e.g., urea, guanidine hydrochloride), which inevitably result in additional pollution, increased costs, and difficulties in further fabrication (Senthilkumar, N., Chowdhury, S. & Sanpui, P. Extraction of keratin from keratinous wastes: current status and future directions. J Mater Cycles Waste Manag 25, 1–16 (2023)).

[0006] As a result, over ten million tons of keratin-rich waste generated annually worldwide from animal husbandry and textile industry are typically disposed through incineration or landfilling instead of being repurposed (Niinimäki, K. et al. The environmental price of fast fashion. Nat Rev Earth Environ 1, 189–200 (2020); Sun, J. et al. Bioinspired Processing of Keratin into Upcycled Fibers through pH-Induced Coacervation. ACS Sustainable Chem. Eng.11, 1985–1994 (2023)). Despite the mistreatment, keratin materials preserve various desirable properties including biodegradability, biocompatibility, mechanical strength, and chemical modificability, thereby endowing them with promising potential for versatile applications including sustainable textiles, wound healing, and tissue engineering (Fang, W., Fan, R., Aranko, A. S., Hummel, M. & Sixta, H. Upcycling of Keratin Wastes in Sustainable Textile Fiber Applications. ACS Sustainable Chem. Eng.11, 14807–14815 (2023); Serag, E., El-Aziz, A. M. A., El-Maghraby, A. & Taha, N. A. Electrospun non-wovens potential wound dressing material based on polyacrylonitrile / chicken feathers keratin nanofiber. Sci Rep 12, 15460 (2022); Zhu, S. et al. Using Wool Keratin as a Structural Biomaterial and Natural Mediator to Fabricate Biocompatible and Robust Bioelectronic Platforms. Adv Sci (Weinh) 10, e2207400 (2023)).

[0007] There remain a need for a scalable and sustainable method to regenerate keratin from keratin-rich sources or keratin-rich waste that relies on a recyclable solute to repurpose the underutilized protein waste into valuable biomaterials. 2 ME1\53235995.v1DOCKET NO.117823-37020 SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention provides a method for preparingregenerated keratin comprising: (a) providing a keratin-rich source; (b) contacting the keratin-rich source with an aqueous solution comprising about 6 M to about 12 M of a denaturing agent and about 0.05 M to about 0.5 M of a reducing agent to obtain a mixture; (c) heating the mixture from room temperature to about 90 °C for about 6 hours to about 72 hours to obtain an extracted mixture; (d) subjecting the extracted mixture to hot filtration to obtain a keratin solution; (e) cooling the keratin solution from about -20 °C to about -4 °C overnight to obtain a cooled keratin solution; (f) centrifuging the cooled keratin solution to obtain a subnatant comprising a denatured keratin gel and a supernatant comprising a solution of the denaturing agent; (g) reconstituting the subnatant comprising the denatured keratin gel in water to yield regenerated keratin; and (h) recycling the supernatant comprising a solution of the denaturing agent for use in steps (a) to (e), thereby preparing the regenerated keratin.

[0009] In one aspect, the keratin-rich source is selected from animal fragments,feather, hair, skin, fingernail, hoof, wool-based textile, or a combination thereof.

[0010] In one aspect, the feather is goose feather.

[0011] In one aspect, the hair is animal hair or human hair.

[0012] In one aspect, the hair is wool.

[0013] In one aspect, the keratin-rich source comprises of alpha-keratin, beta-keratin,or a combination thereof.

[0014] In one aspect, the denaturing agent is Lithium Bromide (LiBr).

[0015] In one aspect, the keratin-rich source is contacted with a solution comprisingabout 6.25 M, about 6.5, about 6.75 M, about 7 M, about 7.25 M, about 7.5, about 7.75 M, about 8 M, about 8.25 M, about 8.5, about 8.75 M, about 8 M, about 8.25 M, about 8.5, about 8.75 M, about 9 M, about 9.25 M, about 9.5, about 9.75 M, about 10 M, about 10.25 M, about 1.5, about 10.75 M, about 11 M, about 11.25 M, about 11.5, or about 11.75 M LiBr.

[0016] In one aspect, the keratin-rich source is contacted with a solution comprisingabout 8 M LiBr.

[0017] In one aspect, the reducing agent is 1, 4-dithiothreitol (DTT).3 ME1\53235995.v1DOCKET NO.117823-37020

[0018] In one aspect, the keratin-rich source is contacted with a solution comprisingabout 0.05 M, about 0.1 M, about 0.15 M, about 0.2 M, about 0.25 M, about 0.3 M, about 0.35 M, about 0.4 M, about 0.45 M, or about 0.5 M DTT.

[0019] In one aspect, the keratin-rich source is contacted with an aqueous solutioncomprising about 0.1 M DTT.

[0020] In one aspect, the recycling in step (h) is a closed-loop recycling.

[0021] In one aspect, the regenerated keratin is alpha-keratin.

[0022] In one aspect, the regenerated keratin is biocompatible and biodegradable.

[0023] 1 In one aspect, the regenerated keratin has a tunable mechanical propertyand has a shape-memory property.

[0024] In one aspect, the present invention provides a method of making a shape-memory object or structure, the method comprising: providing the regenerated keratin prepared according to the above described method; and forming a shape- memory object or structure from a material comprising the regenerated keratin via facile manufacturing.

[0025] In one aspect, the facile manufacturing comprises injection molding, filmcasting, film coating, dip coating, fiber spinning, fiber coating, or 3D printing.

[0026] In one aspect, the shape-memory object or structure comprises smart textiles,biomedical implants, 3D-printed keratin sheets, yarns, threads, polymeric fibers, or fabrics. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Fig.1A is a schematic of Dihydrofolate Reductase (DHFR) representing secondary structure. Fig.1B is a schematic of fibronectin representing secondary and tertiary structures. Fig.1C is a schematic of α-keratin (wool) representing secondary, tertiary and quarternary structures.

[0028] Fig.2A is a turbidity assay of DHFR measured by OD405, suggesting different denaturation capabilities of LiBr, LiCl, and NaBr. Fig.2B is a turbidity assay of fibronectin measured by OD405, suggesting different denaturation capabilities of LiBr, LiCl, and NaBr. Fig.2C is a turbidity assay of α-keratin (wool) measured by OD405, suggesting different denaturation capabilities of LiBr, LiCl, and NaBr. Data are normalized by the highest OD405 values of respective proteins and presented as mean (n = 3).

[0029] Fig.3A is a graph showing the FTIR of the amide I band indicating a gradualloss of secondary structures upon increasing of LiBr concentration in DHFR. Fig. 3B4 ME1\53235995.v1DOCKET NO.117823-37020 is a graph showing the FTIR of the amide I band indicating a gradual loss of secondary structures upon increasing of LiBr concentration in fibronectin. Fig.3C is a graph showing the FTIR of the amide I band indicating a gradual loss of secondary structures upon increasing of LiBr concentration in α-keratin (wool).

[0030] Fig.4 is a graph showing the percentage change of the secondary structures in DHFR in 2 M LiBr, deconvoluted from the amide I band of Raman spectra.

[0031] Fig.5 is a DLS measurement of the hydrodynamic radius of fibronectin with increasing LiBr concentration, indicating an extension of tertiary structure followed by aggregation. Data are presented as mean ± 1.5 s.d. (n = 6).

[0032] Fig.6A is a SEM image of wool prior to denaturation with 8 M LiBr. Fig.6B is a SEM image of wool after denaturation with 8 M LiBr. Scale bars, 50 µm.

[0033] Fig.7 is a graph depicting the DHFR unfolding curve in LiCl solution measured by tryptophan fluorescence (dots) and fitted by two-state model (dash lines). Data are presented as mean ± s.d. (n = 3).

[0034] Fig.8 is a schematic showing the fibronectin conformational changes under different LiBr concentrations as cross-referenced from turbidity, FTIR, and DLS experiments. Fibronectin conformations start from globular protein (0 M), to extended globular protein (~2 M), to aggregation of extended globular protein (4 M-6 M) until aggregation of denatured protein (>6 M).

[0035] Fig.9A is a SEM image of wool prior to 48-hour denaturation with 8 M LiCl. Fig.9B is a SEM image of wool after 48-hour denaturation with 8 M LiCl. Fig.9C is a SEM image of wool prior to 48-hour soak in 7 M NaBr. Fig.9D is a SEM image of wool after 48-hour soak in 7 M NaBr. Fig.9E is a SEM image of feather prior to 48- hour denaturation with 8 M LiBr. Fig.9F is a SEM image of feather after 48-hour denaturation with 8 M LiBr. Scale bars, 50 µm.

[0036] Fig.10A is a schematic for enthalpy-driven denaturation. Fig.10B is a schematic for entropy-driven denaturation.

[0037] Fig.11A is a graph showing the reaction enthalpy during the interactions between DHFR and denaturants (LiBr, urea, and GdnHCl) as measured by ITC. Fig. 11B is a graph showing the reaction enthalpy during the interactions between fibronectin and denaturants (LiBr, urea, and GdnHCl) as measured by ITC. Data are presented as mean ± s.d. (n = 3).

[0038] Fig.12A is a plot of radial distribution function g(r) of oxygen atoms of water molecules around respective cations (solid) and anions (dashed) in 7 M LiBr, LiCl, 5 ME1\53235995.v1DOCKET NO.117823-37020 and NaBr solution. Fig.12B shows the water density at first hydration shell around respective cations (solid) and anions (dashed) in different concentrations of LiBr, LiCl, and NaBr.

[0039] Fig.13 shows the molecular entropy distribution of water molecules in 7M LiBr, LiCl, and NaBr.

[0040] Fig.14A shows the water molecular entropy distribution in LiBr at different concentrations. Fig.14B shows the water molecular entropy distribution in LiCl at different concentrations.

[0041] Figs.15A and 15B are schematics of different ion effects on water dynamics. Fig.15A is a schematic of local effect for LiBr and LiCl. For LiBr and LiCl, high charge density of Li+ion results in localized (bound) water molecules and disrupted water network. Fig.15B is a schematic of global effect for NaBr. For NaBr, changes in water dynamics were observed in a more global manner without break of water networks (Fig.15B).

[0042] Fig.16 shows the water molecular entropy distribution in NaBr at different concentrations.

[0043] Fig.17 is a graph showing the total entropy penalty decrease of protein unfolding caused by water network changes from indirect solute effects of LiBr, LiCl, and NaBr. Data are presented as mean (n = 3) with error bars smaller than point size.

[0044] Fig.18 are graphs showing the decomposition of the total entropy penalty decrease presented in Fig.17.

[0045] Fig.19 is a graph showing the free energy landscape of α-helix peptide (20 amino acids) in pure water and 1, 4, 7 M LiBr.

[0046] Fig.20 is a schematic comparing protein unfolding caused by direct interactions and indirect solute effects.

[0047] Fig.21 is a schematic showing the regeneration protocol of keratin waste with closed-loop recycling of LiBr solution.

[0048] Fig.22A is a plot of keratin content of aggregated gel after separation. Fig. 22B shows the size distribution of denatured keratin in 8 M LiBr solution demonstrating a spontaneous aggregation as temperature decrease from DLS. Fig. 22C shows the size distribution radius of denatured keratin in 8 M LiBr solution as a function of temperature from DLS. 6 ME1\53235995.v1DOCKET NO.117823-37020

[0049] Fig.23 is a plot showing the extraction yield of α-keratin from wool and β- keratin from goose down. Data are presented as mean ± s.d. (n = 7 for α-keratin, n = 3 for β-keratin).

[0050] Fig.24 shows the comparison of TGA profile that shows no major change in the LiBr solution after 5 cycles of extraction.

[0051] Fig.25A is a 3D TG-FTIR of the LiBr solution recycled after keratin separation. Fig.25B is a 2D TG-FTIR of the LiBr solution recycled after keratin separation.

[0052] Fig.26A shows the simple separation of denatured keratin from LiBr solution driven by spontaneous aggregation. Fig.26B shows the homogenous dispersion of denatured keratin in urea solution, which requires an additional step of dialysis to separate. Fig.26C shows that keratin gel separated from LiBr solution exhibits a rapid phase transition as a result of renaturation upon immersion in water.

[0053] Fig.27 shows the facile manufacturing strategies leveraging the advantage of phase transition. Scale bars, 1 cm.

[0054] Fig.28A is a schematic of the alpha-keratin in reduced state and rearrangement of alpha-helices induced by stretch. Fig.28B is a schematic of the alpha-keratin in oxidized state and uncoil of helix upon stretch. Fig.28C is a polarized Raman of reduced keratin sample. The anisotropy of keratin under 50% strain suggests stretch-induced rearrangement of helices.

[0055] Fig.29 shows the cyclic loading of reduced keratin to 50% strain.

[0056] Fig.30 shows the cyclic loading of oxidized keratin to 50% strain.

[0057] Fig.31A is a stress-strain curve of regenerated keratin in reduced state. Fig. 31B is a stress-strain curve of regenerated keratin in oxidated state. Data are presented as mean ± s.d. (n = 4).

[0058] Fig.32A is a graph showing yield stress of regenerated keratin in reduced state and oxidized state. Fig.32B is a graph showing yield strain of regenerated keratin in reduced state and oxidized state. Data are presented as mean ± s.d. (n = 6).

[0059] Fig.33A is a Raman spectra of the amide I band of reduced and oxidized keratin under 50% strain. A more significant change of secondary structures within oxidized sample indicates the uncoiling of helices Dottled lines are peaks indicating specific secondary structures from deconvolution, where alpha and beta peaks are labeled. Fig.33B shows the change of secondary structure percentage under 50% 7 ME1\53235995.v1DOCKET NO.117823-37020 strain (n=3). A more significant decrease of α-helix can be observed with oxidized sample, indicating the uncoiling of helices into metastable β / random structures.

[0060] Fig.34 is a stress-strain curve of reduced and oxidized keratin until fracture. Scale bars, 2 mm.

[0061] Fig.35 is a schematic of the shape memory effect triggered by hydration signal.

[0062] Fig.36 is demonstration of shape memory effect with a badge model and a tensegrity structure. Scale bars: 3 cm.

[0063] Fig.37 is Neonatal Rat Ventricular Myocytes (NRVMs) Form a Beating Monolayer on Keratin 7-Days Post Seeding. Fig.37A is a brightfield image of NRVMs forming a tug of beating myocytes on top of a substrate of fibroblast-looking cells on glass without keratin coating. Scale bars, 200 µm. Fig.37B is a brightfield image of a monolayer of NRVMs beating on keratin. Scale bars, 200 µm. Fig.37C is immunostained images of a monolayer of sparsely and densely seeded NRVMs on glass substrates, treated or untreated with ECM (Geltrex + Fibronectin) and coated or uncoated with keratin, showing clear sarcomere formation and actin fibers. Myocytes stretch more under the presence of keratin alone (no ECM), similar to what they would act with a traditional ECM mixture. Keratin substrate dramatically facilitates adhesion, cell stretching into a rod-shaped format, and size increase as compared to the no keratin substrate without ECM, demonstrating the potency of keratin for tissue engineering. Scale bars, 100 µm. DETAILED DESCRIPTION

[0064] Exemplary embodiments of the present disclosure are illustrated in the drawings, which are illustrative rather than restrictive. No limitation on the scope of the technology, or on the claims that follow, is to be implied or inferred from the examples shown in the drawings and discussed herein.

[0065] Electrolyte is one of the essential carriers of all life forms, where proteins, water, and solutes interplay to support vital activities. Various kinds of direct interactions between proteins / water and solutes (Mazzini, V. & Craig, V. S. J. Volcano Plots Emerge from a Sea of Nonaqueous Solvents: The Law of Matching Water Affinities Extends to All Solvents. ACS Cent. Sci.4, 1056–1064 (2018)) have been observed to explain protein folding, stability, and functions, which then 8 ME1\53235995.v1DOCKET NO.117823-37020 stimulated and enabled rational design, exquisite control, and mass production of protein-based materials. However, a holistic understanding of the trilateral interactions remains elusive for more than a century, largely due to intertwined direct and indirect solute effects (Xie, W. J. & Gao, Y. Q. A Simple Theory for the Hofmeister Series. J. Phys. Chem. Lett.4, 4247–4252 (2013)).

[0066] The current keratin extraction strategies is presented in Table 1. The methods available for protein denaturation include applying heat, UV radiation, microwave, etc. Other methods include hydrolysis using an acid, base or enzyme. Heavy metal or alkaloids such as Hg, Ag, SDS, etc have also been used towards protein denaturation. Strong solvents such as formic acid, ionic liquids have also been employed in such methods. Organic denaturing agents include urea, guanidium (Gdm).

[0067] The above mentioned denaturing methods are expensive and often time- consuming. Further other problems of extracting protein, such as keratin using the above mentioned denaturing methods include difficulty during extraction and regeneration process.

[0068] In addition, organic denaturing agents such as urea interfere with some of the most widely used analytical tools such as FTIR and Raman due to the interaction with backbone carbonyl group (Thirumali et al., J. A. Chem. Soc., 2007). Also in methods of protein extraction using urea or Gdm, includes additional time-consuming steps like dialysis.

[0069] Thus, the currently available methods contribute to waste and pollution and results in non-recovery of denaturing agents.

[0070] Table 1: Summary of Current Keratin Extraction Strategies.9 ME1\53235995.v1DOCKET NO.117823-3702010 ME1\53235995.v1DOCKET NO.117823-3702011 ME1\53235995.v1DOCKET NO.117823-3702012 ME1\53235995.v1DOCKET NO.117823-37020

[0071] Significance of entropy-driven mechanism in regeneration of protein waste: Millions of tons of animal protein waste are generated yearly around the world in textile industry and livestock husbandry (Timorshina et al. Sustainable Applications of Animal Waste Proteins. Polymers 14, 1601 (2022)). For instance, more than 10 kg CO2-eq / kg is emitted during the production of 1 kg fine-grade wool (Peri et al. Sustainability 12, 3077 (2020)), yet 85% of textiles in the US end up landfilled or incinerated (Sandin et al. Journal of Cleaner Production 184, 353–365 (2018)). Similar situation can also be found in farming industry, where more than 40 million tons of animal body parts are generated annually (Peng et al. ACS Sustainable Chem. Eng.7, 9727–9736 (2019)), with the vast majority going to incineration and burial. As a structural protein that forms epidermal and epidermal appendages of animals (Wang et al. Progress in Materials Science 76, 229–318 (2016)), keratin is the major composition of wool, hair, feathers, hoofs and skin, contributing 50%-90% of the aforementioned waste (Shurson et al. Sustainability 12, 7071 (2020)). 13 ME1\53235995.v1DOCKET NO.117823-37020 However, the inherent stability of keratin and its complex extraction process continue to pose significant challenges in utilizing keratin-rich resources, with the high cost of extraction and associated pollution being the primary obstacles.

[0072] To address this problem and regenerate keratin-rich waste, a variety of strategies haven been developed and studied over the past few decades as shown in Table 1. Despite their varying approaches, a common issue is the single-use of chemicals, solvents, or enzymes during extraction, which inevitably increases costs and leads to secondary pollution. This is particularly problematic for the valorization of inexpensive and low-risk waste like proteins, for which incineration and landfill could always be alternative solutions. For example, traditional approaches with organic denaturing agents (Ozaki et al. Materials Science and Engineering: C 42, 146–154 (2014)), Deb-Choudhury et al. Methods Enzymol 568, 279–301 (2016)), Kadirvelu et al. Sci Rep 6, 36558 (2016)), Ramya et al. Process Biochemistry 90, 223–232 (2020)) requires additional steps of dialysis and lyophilization after extraction due to the direct binding between organics and proteins, while other methods including acidic / basic hydrolysis (Earland et al. Biochim Biophys Acta 17, 457–461 (1955)), Cardamone, Journal of Molecular Structure 969, 97–105 (2010)), Zhang et al. Integrated Ferroelectrics 160, 169–174 (2015)), Bhat et al. Ultrasonics Sonochemistry 71, 105368 (2021)), microbial / enzymatic degradation (Zhang et al. Journal of Cleaner Production 192, 433–442 (2018)), Peng et al. Microbial Cell Factories (2019)), Su et al. Macromol Biosci 20, e2000073 (2020)), Lai et al. Biotechnol Biofuels Bioprod 16, 59 (2023)), microwave irradiation (Zoccola et al. Textile Research Journal 82, 2006–2018 (2012)), Bertini et al. Polymer Degradation and Stability 98, 980–987 (2013)), Chen, et al. Chemical Engineering and Processing: Process Intensification 87, 104–109 (2015)), and steam explosion (Tonin et al. Biomacromolecules 7, 3499–3504 (2006)), Zhang et al. ACS Sustainable Chem. Eng.3, 2036–2042 (2015)) would also result in irreversible destruction of protein integrity. Furthermore, even the more sustainable approach using ionic liquids (Xie et al. Green Chem.7, 606–608 (2005)), Lovejoy et al. Anal. Chem.84, 9169–9175 (2012)), Ji et al. Separation and Purification Technology 132, 577–583 (2014)), Apostolidou, ChemistryOpen 9, 695–702 (2020)), Fang et al. ACS Sustainable Chem. Eng.11, 14807–14815 (2023)) necessitates extra precipitation steps in water or ethanol for separation, creating challenges in post-fabrication and ion recovery. From a mechanistic perspective, the direct interactions between 14 ME1\53235995.v1DOCKET NO.117823-37020 denaturants and proteins pose unavoidable problems of separation and chemical wastage, which cannot be bypassed using our current understanding of denaturation process.

[0073] Notably, the use of lithium bromide (LiBr) in protein extraction has been of longstanding interest. Beside keratin denaturation, LiBr has also been employed in the regeneration of other biomasses such as silk fibroin (Feng et al. ACS Sustainable Chem. Eng.5, 6227–6236 (2017)), cellulose (Yang et al. Cellulose 21, 1175–1181 (2014)), and chitosan (Kim et al. Cellulose 25, 2615–2628 (2018)), where less interference from disulfide crosslinking occurs. However, the extraction and post-processing steps involving LiBr share many similarities with organic denaturants, including dialysis and lyophilization / evaporation. Some studies have also explored mixing LiBr with other organic or oxidative denaturants to enhance extraction efficiency (Zeng et al. Advanced Materials Research 881–883, 551–555 (2014)), Wang et al. ACS Sustainable Chem. Eng.12, 6003–6012 (2024)). Recent approaches are beginning to consider alternative methods for phase separation, such as introducing salts (Cera et al. Nat Mater 20, 242–249 (2021)) into the mixture or lowering the pH to induce precipitation (Sun et al. ACS Sustainable Chem. Eng. 11, 1985–1994 (2023)), but the recycling of LiBr has not yet been considered due to an insufficient understanding of the denaturation mechanism itself.

[0074] Unraveling the entropy-driven mechanism of denaturation not only provides insights into the complex interactions between proteins, water, and solutes, but it also reshapes our perception of the role that LiBr plays in the extraction of protein- based materials. When direct interactions no longer play the major role in protein denaturation, it opens the possibility for a bottom-up refinement of the entire regeneration process. This includes leveraging spontaneous aggregation for simple separation, enabling the closed-loop recycling of LiBr, and utilizing the rapid phase transition of denatured keratin gel for facile manufacturing.

[0075] The inventors have solved the above problems associated with protein extraction by identifying a method of protein denaturation for regenerating sustainable keratin from keratin-rich sources or keratin-rich wastes, such as, but not limited to wool textiles and from poultry waste such as hair, feather. The denaturation process described in the methods of the disclosure is achieved via indirect interaction and includes a closed-loop recycling for achieving true sustainability. Aggregation of denatured keratin and unchanged concentration of the 15 ME1\53235995.v1DOCKET NO.117823-37020 denaturant, such as LiBr enables recycle and recovery for multiple cycles. Further no change in LiBr solution composition was observed in the methods of the disclosure.

[0076] Although water itself is a well-established environment, concentrated ions may affect its property including entropy drastically. Pure water or low salt concentrations, such as 4 M Li Br, 8 M LiCl can result in increasing water network entropy and increase the entropy penalty leading to a hard separation of protein. On the contrary, concentrated ions, such as 8M LiBr can disturb water network and reduce the entropy penalty of water-protein interaction. This is favorable in the methods of the disclosure as it follows an easy entropy-driven protein denaturation mechanism.

[0077] In the present disclosure, the inventors show how solutes can indirectly manipulate protein conformations through changing water networks as quantified by entropy. Choosing lithium bromide (LiBr) as a denaturant, the universal protein denaturation capability is unveiled underpinned by the entropy-driven mechanism given absent direct ion-protein interactions.

[0078] Further, a scalable strategy for protein waste revalorization, distinguished by the closed-loop recycling of denaturants, straightforward protein separation, and simplified post-processes, all enabled by the entropy-driven denaturation via LiBr is presented herein.

[0079] This disclosure relates in part to a sustainable regeneration of keratin from keratin-rich sources, featuring a closed-loop recycling of a denaturing agent, such as Lithium Bromide (LiBr), alongside diverse manufacturing pathways. The resulting keratin material demonstrates tunable mechanical properties, shape-memory capability, and has promising potential in a variety of applications.

[0080] Provided herein are methods for regenerating keratin and uses thereof for fabricating biocompatible, shape-memory polymeric fibers, threads, yarns, fabrics, and shape-memory objects from natural materials.

[0081] The methods of the present invention are useful in various applications. By recapitulating the hierarchical organization, conformational change and denaturation capabilities of monovalent ion pairs, such as LiBr, the keratin regenerated by the methods of the disclosure serves as a desirable material for use in facile manufacturing of a shape-memory object or a structure. 16 ME1\53235995.v1DOCKET NO.117823-37020

[0082] In the following description, for purposes of explanation, specific numbers, materials and configurations are set forth in order to provide a thorough understanding of the invention. It will be apparent, however, to one having ordinary skill in the art that the invention may be practiced without these specific details. In some instances, well-known features may be omitted or simplified so as not to obscure the present invention. Furthermore, reference in the specification to phrases such as “one embodiment”, “an embodiment”, “an example embodiment” or “an exemplary embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the invention and could possibly be included in multiple different embodiments. The appearances of phrases such as “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment.

[0083] In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this invention.

[0084] In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

[0085] I. Definitions

[0086] Applicant specifically incorporates the entire contents of all cited references in this disclosure. Further, when an amount, concentration, or other value or parameter is given as either a range or a list of upper values and lower values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or value and any lower range limit or value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the present disclosure be limited to the specific values recited when defining a range.

[0087] The indefinite articles “a” and “an”, as used herein should be understood tomean “at least one,” unless clearly indicated to the contrary. 17 ME1\53235995.v1DOCKET NO.117823-37020

[0088] The phrase “and / or”, when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, “A, B, and / or C” indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase “and / or” may be used interchangeably with “at least one of’ or “one or more of’ the elements in a list.

[0089] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0090] “Alpha-keratin” or “α-keratin” is an a-helical fibrous protein found in higher animals such as mammals, birds and reptiles. Alpha-keratin is the primary component of hairs, horns, nails and the epidermal layer of the skin. The secondary structure of natural alpha-keratin is composed predominantly of alpha-helices, which form coiled- coil dimers in strands along the fiber axis. Dimers are typically formed by one neutral- basic keratin (type II) fiber and one acidic keratin (type I) fiber. In turn, these dimers coil together in an antiparallel manner to form tetramers. When tetramers are connected from head to tail, they are known as “protofilaments.” The protofilaments pair to form protofibrils. Protofibrils self-assemble to form a bundle called an “intermediate filament” (“IF”), or “keratin intermediate filament” (“KIF”), with a diameter of about 70-110 A is formed. Keratin intermediate filaments further assemble into micro filaments with a diameter of about 0.1-0.4 mih and multiple micro filaments of keratin form macrostructures such as animal hair with a diameter of about 50 pm (see, R. A. Quinlan et al, Ann. N. Y. Acad. Sci.1985, 455, 282-306, H. Thomas et al. Int. J. Biol. Macromol.1986, 8, 258-264 and C. Popescu et al. Chem. Soc. Rev., 2007, 36, 1282-1291).

[0091] As used herein, a “reducing agent” is a compound that cleaves the disulfide bonds (S-S) present in the keratin-rich source, by forming two thio groups (SH) and prevents the protein from aggregating. Exemplary reducing agents include, but are not limited to 1, 4-dithiothreitol (DTT), 2-Mercaptoethanol (BME), dithioerythritol (DTE), Tris (2-Carboxyethyl) phosphine hydrochloride) (TCEP) and L-glutathione (GSH). In some embodiments, a reducing agent is 1, 4-dithiothreitol (DTT).

[0092] As used herein, the term “powder” refers to a dry, bulk solid including fine 18 ME1\53235995.v1DOCKET NO.117823-37020 particles that flow freely when shaken or tilted.

[0093] As used herein, the term “suspension” is a heterogeneous mixture that contains solid particles sufficiently large for sedimentation. The particles may be visible to the naked eye or under a microscope, usually must be larger than 1 micrometer, and will eventually settle. Suspensions are heterogeneous mixtures in which the solid particles do not dissolve, but get suspended throughout the bulk of the solvent, left floating around freely in the medium. The internal phase (solid) is dispersed throughout the external phase (fluid) through mechanical agitation.

[0094] As used herein, the term “solution” is a homogeneous liquid mixture including two or more substances, the minor component (the solute) which is uniformly distributed within the major component (the solvent).

[0095] As used herein, the term “two-phase” is a mixture of two immiscible solutions in which the boundary of separation of the two solutions is visible.

[0096] As used herein, the terms “fiber” and “polymeric fiber” are used herein interchangeably, and both terms refer to fibers having micron, submicron, and nanometer dimensions and including alpha-keratin intermediate filaments. In some embodiments, a polymeric fiber includes a plurality of alpha-keratin protofibrils and / or intermediate filaments.

[0097] As used herein, “hair” includes human hair and hair of other animals (e.g., mammals) including, but not limited to wool and fur.

[0098] The term “closed-loop recycling” as used herein refers to use and recovery of a denaturing agent, such as Lithium Bromide (LiBr) for reuse in a process of regenerating keratin from keratin-rich sources as described herein.

[0099] As used herein, “shape-memory” is a property of certain smart materials to return from a deformed state to their original (e.g., fixed) shape induced by an external stimulus (trigger). Triggers of a shape-memory effect include, but are not limited to light, moisture, and temperature change.

[0100] As used herein, the term “3D printing” refers to using a 3D printing device or 3D printing system to manufacture an object, and other additive manufacturing methods.

[0101] II. METHODS OF THE INVENTION

[0102] A. Methods of regenerating keratin from keratin-rich sources.

[0103] As described above, the present invention is based, at least in part, on the discovery of methods of regeneration of keratin from keratin-rich sources, that preserve the hierarchical organization of keratin and has long-range molecular order 19 ME1\53235995.v1DOCKET NO.117823-37020 and shape-memory properties using a closed-loop recycling of a denaturing agent.

[0104] The combination of conformational change and denaturation capability of monovalent ion pairs, such as LiBr, and recapitulating the native hierarchical organization of keratin allows for the scalable fabrication of strong and hierarchically structured shape-memory fibers and 3D printed scaffolds with potential applications in bioengineering and smart textiles.

[0105] Accordingly, in one aspect, the present invention provides a method of regenerating keratin comprising of steps a-h respectively.

[0106] Step (a):

[0107] The method begins with providing a keratin-rich source. Such keratin-rich sources can include animal fragments, feather, hair, skin, fingernail, hoof, wool-based textile, or a combination thereof.

[0108] Hair suitable for use in the methods of the invention may be human hair or animal hair. In one embodiment, the hair is animal hair. In one embodiment, the animal hair is wool, for e.g., sheep wool, goat wool, alpaca wool, or rabbit fur.

[0109] In one embodiment, the feather is goose feather, such as goose down.

[0110] The keratin-rich source may be pre-processed or pre-treated for use in the said methods. For example, as described in the Examples, Angora goat wool was obtained and washed using, e.g., ethanol, to remove oils and other debris. Other agents suitable to wash hair prior to preparing a hair powder include, for example, any suitable solvent such as petroleum, ether, or acetone. The washed wool fibers were cut into segments and the wool segments were ground into micron- sized particles. Other methods for grinding the hair into a powder include, for example, a pestle and mortar.

[0111] In some embodiments, hair, e.g., wool, is washed in ethanol for about 1 to about 48 hours, about 1 to about 48 hours, about 1 to about 48 hours, about 1 to about 48 hours, about 2 to about 48 hours, about 3 to about 48 hours, about 4 to about 48 hours, about 5 to about 48 hours, about 6 to about 48 hours, about 7 to about 48 hours, about 8 to about 48 hours, about 9 to about 48 hours, about 10 to about 48 hours, about 11 to about 48 hours, about 12 to about 48 hours, about 13 to about 48 hours, about 14 to about 48 hours, about 15 to about 48 hours, about 16 to about 48 hours, about 17 to about 48 hours, about 18 to about 48 hours, about 19 to about 48 hours, about 20 to about 48 hours, about 21 to about 48 hours, about 22 to about 48 hours, about 23 to about 48 hours, or about 24 to about 48 hours, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 20 ME1\53235995.v1DOCKET NO.117823-37020 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or about 48 hours. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[0112] In some embodiments, the washed hair is rinsed, e.g., in water, and allowed to dry, e.g., at room temperature. The washing and rinsing of the hair may be performed once, or may be repeated two or more times.

[0113] Segments or fibers of the washed hair may be prepared using any suitable method.

[0114] For example, the hair, e.g., wool fibers, may be cut, e.g., manually, into segments that are approximately about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm in length, about 9 mm, about 10 mm, or any other suitable size. Values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[0115] Step (b):

[0116] The keratin-rich source, such as, but not limited to hair, wool, feather is contacted with an aqueous solution of a denaturing agent and an aqueous solution of a reducing agent.

[0117] In some embodiments, the keratin-rich source is contacted with an aqueous solution comprising about 6 M to about 12 M of a denaturing agent and about 0.05 M to about 0.5 M of a reducing agent to obtain a mixture.

[0118] Denaturing Agent:

[0119] In some embodiments, the denaturing agent is a monovalent ion pairs, such as Lithium Bromide (LiBr) and Lithium Chloride (LiCl).

[0120] In a specific embodiment, the denaturing agent is Lithium Bromide (LiBr). LiBr induces a reversible and concentration dependent liquid phase transition of hard alpha-keratin and is used as a mild denaturing agent for the extraction and regeneration of keratin from keratin-rich sources, such as hair.

[0121] Also, LiBr has the second largest solubility in all common solvents (e.g., water, methanol, ethanol, etc.) and stable chemical properties as contrast to lithium iodide (LiI). Besides, LiBr has been discovered to dissolve silk fibroin by von Weimarn in 1925 (Ambrose, E. J., Bamford, C. H., Elliott, A. & Hanby, W. E. Water-Soluble Silk: an α-Protein. Nature 167, 264–265 (1951)), followed by keratin contraction study in LiBr solution by Feughelman et al. in 1962 (Feughelman, M., Haly, A. R. & Mason, P. Contraction of Keratin Fibres in Aqueous Lithium Bromide. Nature 196, 957–958 21 ME1\53235995.v1DOCKET NO.117823-37020 (1962)). The absence of reliable mechanisms has been persistent until nowadays and largely limited its further industrial applications.

[0122] The inventors have systematically explored LiBr’s effects on protein structure and denaturation through comparison with both lithium chloride (LiCl) and sodium bromide (NaBr). The universal denaturation capability of LiBr has been confirmed by testing three proteins spanning all different levels of structure complexity as shown in the Examples.

[0123] As described in the Examples, LiBr has the strongest capability of unfolding (denaturing) proteins, followed by LiCl. NaBr cannot denature keratin.

[0124] Traditional denaturation methods using enthalpy-driven denaturants, such as, urea or guanidine hydrochloride (GdnHCl) involve direct protein-denaturant interactions, lead to difficulties in protein separation and further limits protein regeneration. Such enthalpy-driven denaturants also present a major problem contributing to waste and pollution from single-used organic chemicals and results in complexity of fabrication.

[0125] On the other hand, the entropy-driven protein denaturation via LiBr and the absence of direct protein-denaturant interactions enables the sustainable regeneration of keratin from keratin-rich sources.

[0126] The methods of the invention also features closed-loop LiBr recycling, simple protein separation, and facile manufacturing that are described herein.

[0127] The concentration of LiBr in the aqueous solution may be, for example, about 6 M to about 12 M, about 6.25 M to about 11.75 M, about 6.5 M to about 11.5 M, about 6.75 M to about 11.25 M, about 7 M to about 11 M, about 7.25 M to about 10.75 M, about 7.5 M to about 10.5 M, about 7.75 M to about 10.25 M, about 6 M to about 11 M, about 6.25 M to about 10.75 M, about 6.5 M to about 10.5 M, about 6.75 M to about 10.25 M, about 7 M to about 9 M, about 7.25 M to about 9.75 M, about 7.5 M to about 9.5 M, about 7.75 M to about 9.25 M, about 7 M to about 10 M, about 7.25 M to about 9.75 M, about 7.5 M to about 9.5 M, about 7.75 M to about 9.25 M, e.g., about 6 M, about 6.25 M, about 6.5, about 6.75 M, about 7 M, about 7.25 M, about 7.5, about 7.75 M, about 8 M, about 8.25 M, about 8.5, about 8.75 M, about 8 M, about 8.25 M, about 8.5, about 8.75 M, about 9 M, about 9.25 M, about 9.5, about 9.75 M, about 10 M, about 10.25 M, about 1.5, about 10.75 M, about 11 M, about 11.25 M, about 11.5 M, or about 11.75 M, or about 12 M. In some embodiments, the keratin-rich source is contacted with an aqueous solution of about 8 M LiBr. Ranges 22 ME1\53235995.v1DOCKET NO.117823-37020 and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[0128] Reducing Agent:

[0129] In some embodiments, the reducing agent is any agent that inhibit oxidative damage and aggregation of the proteins, such as keratin in solution. Exemplary reducing agents include, but are not limited to, 1, 4-dithiothreitol (DTT), 2- Mercaptoethanol (BME), dithioerythritol (DTE), Tris (2-Carboxyethyl) phosphine hydrochloride) (TCEP) and L-glutathione (GSH). In some embodiments, the reducing agent is DTT. DTT breaks down the dense disulfide matrix of keratin.

[0130] The concentration of DTT in the aqueous solution may be, for example, about 0.05 M, about 0.1 M, about 0.15 M, about 0.2 M, about 0.25 M, about 0.3 M, about 0.35 M, about 0.4 M, about 0.45 M, or about 0.5 M DTT. In some embodiments, the concentration of DTT in the aqueous solution is about 0.1 M DTT. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[0131] Step (c):

[0132] In some embodiments, the mixture from step (b) is heated from room temperature to about 90 °C for about 6 hours to about 72 hours to obtain an extracted mixture. In some specific embodiments, the mixture from step (b) is heated from room temperature to about 90 °C for about 36 hours to about 48 hours. The heating is applied in this step in order to accelerate the denaturation reaction kinetics and also to prevent aggregation during extraction.

[0133] Step (d):

[0134] After extraction, the extraction mixture from step (c) is subjected to hot filtration, e.g., filtration performed between room temperature and about 90 °C to remove insoluble residues and to obtain a keratin solution. The hotter the extraction mixture, the easier the filtration process.

[0135] Step (e):

[0136] The keratin solution from step (d) is then cooled overnight from about -20 °C to about -4 °C to obtain a cooled keratin solution. This cooling step is performed for better aggregation of the desaturated keratin.

[0137] Step (f):

[0138] This step includes centrifugation of the cooled keratin solution, such as ultracentrifugation, e.g.300 to 3000 rcf at a lower temperature, such as about 4 °C to 23 ME1\53235995.v1DOCKET NO.117823-37020 about room temperature. Upon centrifugation, a subnatant comprising a denatured keratin gel and a supernatant comprising a solution of the denaturing agent, such as LiBr are separated out.

[0139] Step (g):

[0140] The subnatant comprising the denatured keratin gel from step (f) is then reconstituted in water to obtain the regenerated keratin.

[0141] Step (h):

[0142] Since a denaturing agent, such as LiBr does not achieve denaturation via direct interactions, the resulting supernatant comprising a solution of the denaturing agent, such as LiBr from step (e) can therefore be continuously recycled in a closed-loop process, which not only eliminates pollution from waste organics, but also significantly reduces the consumption and cost of denaturants.

[0143] In the methods of the present application, a salt solution, such as a NaCl salt solution, a phosphate salt solution, calcium chloride (CaCl) solution, magnesium chloride (MgCl) solution, a sulfate salt solution, or a combination of any of aforementioned, is not included to, e.g., form a two phase solution, in any of the steps following the addition of the LiBr and reducing agent.

[0144] In some embodiments, the keratin regenerated from the methods of invention can be alpha-keratin, beta-keratin, or a combination thereof.

[0145] In some embodiments, the methods described herein are sustainable and scalable.

[0146] In some embodiments, the regenerated keratin is biocompatible and biodegradable.

[0147] In some embodiments, the regenerated keratin has tunable mechanical properties and shape-memory capability and holds promise for applications such as smart textiles or next-generation biomedical implants.

[0148] B. Methods of making shape-memory object or structure.

[0149] The keratin regenerated from the above described method of invention maintains the helical nature and therefore amenable for use in facile manufacturing process.

[0150] In one embodiment, the invention relates to a method of making a shape- memory object or structure, the method comprising providing a regenerated keratin and forming a shape-memory object or structure from a material comprising the regenerated keratin via facile manufacturing. 24 ME1\53235995.v1DOCKET NO.117823-37020

[0151] In one embodiment, the facile manufacturing comprises injection molding, film casting, film coating, dip coating, fiber spinning, fiber coating, or 3D printing.

[0152] The invention also includes shape-memory object or structure, such as, but not limited to, smart textiles, biomedical implants, 3D-printed keratin sheets, shape- memory yarns, shape-memory threads, shape-memory polymeric fibers, or shape- memory fabrics.

[0153] The shape-memory polymeric fibers including alpha-keratin protofibrils and intermediate filaments may also be used to prepare shape-memory threads, shape- memory yarns and shape-memory fabrics using any known methods for the same. The shape-memory materials prepared according to the methods of the invention are superior to other shape-memory materials such as synthetic shape-memory materials in many aspects.

[0154] By recapitulating the native hierarchical organization and architecture, the shape-memory yarns, shape-memory threads, shape-memory polymeric fibers, and shape-memory fabrics provide a high level of freedom of manipulating, self-healing, responsiveness to various stimuli such as moisture, and adaptability to various environments and conditions.

[0155] More importantly, the shape-memory object or structure prepared according to the methods of the current invention are biocompatible and biodegradable, therefore having great potential for massive and environment-friendly industrial production.

[0156] The shape-memory object or structure prepared according to the methods of current invention are smart materials and can be widely used in fields such as clothing, medical and cosmetic surgeries, drug delivery, building material production, and manufacturing of materials that require high adaptability to extreme environments.

[0157] Materials & Methods

[0158] Escherichia coli dihydrofolate reductase synthesis: Cell culture. E. coli dihydrofolate reductase (DHFR, UniProt: P0ABQ4) (wild-type or W30C mutant) was overexpressed in BL21(DE3) E. coli cells. The plasmid was synthesized by GenScript Biotech with pET-28a(+) vector and C-terminus 6xHis-tag. All the cell culture were conducted in Terrific Broth (TB) medium (BD Difco™), supplemented with 0.4% (v / v) glycerol (RPI), 4 g L-1 glucose (Sigma-Aldrich), 25 mM MOPS (Sigma-Aldrich, pH=7.2), 0.4 mM citric acid (VWR Chemicals BDH®), 40 µl L-11% (w / v) ferric ammonium citrate (AMRESCO) solution, and 50 µg ml-1kanamycin (Millipore-Sigma). All the concentrations denoted here are final concentrations.2% (v / v) frozen cell stock 25 ME1\53235995.v1DOCKET NO.117823-37020 solution of BL21(DE3) E. coli cells transformed with DHFR (wild-type or W30C mutant) plasmid was used for a small overnight (~12 h) culture at 37 °C with shaking at 250 rpm. 2% (v / v) overnight culture solution was used to start the production culture, initially at 37 °C with shaking at 250 rpm. After 3.5 hours at which the cell growth started the mid-log phase with an optical density of around 0.6 at 600 nm, isopropyl β-D-thiogalactopyranoside (IPTG, RPI) was added to a final concentration of 100 µM to induce the protein overexpression. The temperature was decreased to 18 °C, with shaker speed kept as 250 rpm. The cell pellets were harvested after 12-hour low- temperature protein overexpression through centrifugation.

[0159] Protein purification: Three different buffers were used in DHFR protein purification: Lysis buffer [100 mM bicine (Sigma-Aldrich), 5 mM imidazole (Millipore- Sigma OmniPur®), 250 mM NaCl (VWR Chemicals BDH®), pH=9]; Buffer A [20 mM Tris (J.T. Baker™), 5 mM imidazole, 250 mM NaCl, pH=8.5]; Buffer B [20 mM Tris, 50 mM imidazole, 250 mM NaCl, pH=8]. Every 1 g cell pellet (from 50 ml cell culture solution) was resuspended in 1.5 ml lysis buffer, supplemented with 0.01% (v / v) Nuclease (Millipore-Sigma Benzonase®), 1% (v / v) Protease Inhibitor Cocktail Set II (Millipore-Sigma Calbiochem®), and 1 mg ml-1 lysozyme (Thermo Fisher Scientific). All the concentrations denoted here are final concentrations. The resuspended solution was then sonicated, centrifuged with supernatant collected. cOmplete™ His- Tag Purification Resin (Roche) was used to further isolate DHFR protein from supernatant in gravity flow columns (Bio-Rad). Buffer A was first used to wash away non-bound proteins and other cell debris, followed by elution of Buffer B, where DHFR was washed out and collected. Only the fractions with A280 / A260 > 1.7 were pooled based on the absorbance measurement with a NanoDrop spectrophotometer (Thermo Fisher Scientific). DHFR was then washed into 20 mM Tris buffered saline (pH=7.0, with 250 mM NaCl) and concentrated into a final concentration of 3 mg ml-1using ultrafilter (Sartorius Vivaspin®). Mass spectrometry (Bruker Impact II q-TOF) was used to confirm that DHFR (both wild-type and W30C mutant) was successfully synthesized and purified, after which protein solution was aliquoted and stored in -70 °C.

[0160] Turbidity assay: Turbidity measurements at OD405 were performed using a Biotek PowerWave HT 340 microplate reader. For each condition, protein solutions was mixed with solutions of LiBr, LiCl, and NaBr (Sigma-Aldrich) to a final concentration of 0.4 mg ml-1for DHFR or 0.1 mg ml-1for human fibronectin (BD 26 ME1\53235995.v1DOCKET NO.117823-37020 Biosciences), with a total volume of 100 µl in a 96-well plate. Samples were then immediately placed into the microplate reader and OD405 was continuously measured for 1 h at room temperature. For keratin, 80 mg of cleaned wool (R. H. Lindsay Wool Company) was soaked into 4 ml of salt solutions with 0.1 M DTT (Sigma-Aldrich) in quartz cuvettes, then heated to 70 °C to accelerate the denaturation kinetics. Samples of the solutions were taken at 24 h and 48 h, added into 96-well plate, and cooled at room temperature before OD405 measurement. Photos recording the condition of the solution were taken after cooling the cuvette at room temperature for 1h.

[0161] FTIR and TG-FTIR: All FTIR measurements were conducted using a Nicolet iS50 FTIR spectrometer. Protein solutions with final concentrations of 1 mg ml-1DHFR, 0.5 mg ml-1 fibronectin, and 1 mg ml-1keratin (α-keratin from Angora wool, R. H. Lindsay Wool Company; β-keratin from goose down, Dream Solutions USA), respectively, were prepared with a gradient of LiBr concentrations ranging from 0 to 8 M, then stabilized for at least 3 h prior to measurements. During acquisition, background information was initially recorded by applying pure LiBr solutions onto the ATR crystal, which was then replaced by the corresponding protein solutions, with a total of 64 scans collected for each sample. Solid samples, including regenerated keratins and raw materials, were measured using the same ATR-FTIR setup. Gas phase FTIR coupled with TGA was collected using the same equipment connected to the Discovery TGA 550. Data analysis and background subtraction were carried out with OMNIC v.9.2.86 software.

[0162] Raman and polarized Raman: Raman experiments were performed with a Horiba LabRam HR Evolution Raman confocal system (633nm excitation, 50x, 0.5NA, 13mW). For DHFR experiment, a customized imaging chamber made by sandwiching a 1 mm thick PDMS (Sylgard™ 184, Dow Corning) well between two glass slides was loaded with a solution of 3 mg ml-1DHFR and 2 M LiBr. Each spectrum was collected with 600 gr mm-1, 180 s acquisition time and 2 accumulations. For solid samples, 1800 gr mm-1, 60 s acquisition and 4 accumulations were used. For polarized Raman, an additional increment of 6° per step were applied with the highest intensity tracked around 1650 cm-1. Data collection and analysis were carried out with Labspec v.6.5.1 software.

[0163] Dynamic light scattering: DLS experiments were performed using a Malvern Zeitasizer Pro system. Fibronectin in LiBr solutions were prepared with a final concentration of 0.5 mg ml-1, stabilized for at least 3 h, and filtered through a 0.45 µm 27 ME1\53235995.v1DOCKET NO.117823-37020 PTFE filter (VWR) before transferred into 40 µl cuvettes (Malvern Panalytical) for measurement. Keratin solutions after extraction were diluted to approximately 1 mg ml-1, filtered, then transferred into 1 ml cuvettes (Malvern Panalytical). Samples were heated to 70 °C for 30 min, then stabilized at designated temperature for 10 min before measurement. A reversible transition between smaller diameters at high temperature and larger diameters at low temperature can be observed within individual samples. Data collection and analysis were carried out with ZS Xplorer v.3.0.0.53 software.

[0164] Scanning electron microscopy: Samples were mounted on a 12.5 mm diameter SEM stub covered with carbon tape, then sputtered-coated with Pt / Pd with an EMS 150T ES sputter coater with 10 nm thickness. SEM images were taken with a Zeiss Gemini 360 field emission scanning electron microscope with an electric high tension of 3 kV and SE2 detector.

[0165] Isothermal titration calorimetry: ITC experiments were conducted using a Malvern Panalytical MicroCal VP-ITC. During the experiments, the sample chamber was filled with 1.8 ml of 1 mg ml-1DHFR (W30C mutant) solution or 0.5 mg ml-1fibronectin solution, while the syringe was loaded with 100 µM solution of LiBr, urea, or GdnHCl (Sigma-Aldrich). Corresponding control experiments were performed by titrating milli-Q water into the same concentration of protein solution to measure and subtract the dilution heat of proteins. Additionally, parallel control groups were also performed by titrating 100 µM ligands into a chamber filled with milli-Q water to measure the dilution heat of ligands, which were found to be significantly smaller than the detectable binding enthalpy between proteins and ligands. Each individual run comprised titrating 7 µl ligand solution 25 times into the chamber, with a duration of 12 s, and interval of 240 s, a filter period of 2 s, a stir speed of 309, and temperature controlled at 30 °C. Data collection and analysis including baseline adjustment and integration of enthalpy were carried out with VPViewer2000 v.1.29.1 software.

[0166] Molecular dynamics simulations: General simulation setup. All the molecular dynamics simulations were conducted with NAMD 2.14 (Phillips, J. C. et al. Scalable molecular dynamics on CPU and GPU architectures with NAMD. J Chem Phys 153, 044130 (2020)) and analyzed and visualized with VMD 1.9.3 (Humphrey, W., Dalke, A. & Schulten, K. VMD: Visual molecular dynamics. Journal of Molecular Graphics 14, 33–38 (1996) , unless otherwise denoted. Amber ff14SB force field (Maier, J. A. et al. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015)) were used for atomistic 28 ME1\53235995.v1DOCKET NO.117823-37020 protein simulation, combined with TIP3P water model (Price, D. J. & Brooks, C. L. A modified TIP3P water potential for simulation with Ewald summation. J Chem Phys 121, 10096–10103 (2004)) for explicit solvation and Li / Merz ion parameters (Li, P., Song, L. F. & Merz, K. M. Jr. Parameterization of Highly Charged Metal Ions Using the 12-6-4 LJ-Type Nonbonded Model in Explicit Water. J. Phys. Chem. B 119, 883–895 (2015)) for monovalent ions. All initial configurations of simulation box were prepared using tLEaP program (Case, D. A. et al. The Amber biomolecular simulation programs. Journal of Computational Chemistry 26, 1668–1688 (2005)), followed by minimization, heating, constant pressure (NPT) equilibration, and constant volume (NVT) equilibration. All the production simulations were run under constant particle number, constant volume, and constant temperature (i.e., canonical ensemble (NVT)). Periodic boundary condition was applied to prevent the boundary effect. Langevin thermostat with damping coefficient of 1 ps-1was used to control simulation temperature as 298 K, without coupling hydrogen atoms. Electrostatic and van der Waals interactions were cut off beyond 12 Å. Particle Mesh Ewald (PME) method was employed to deal with long-range electrostatic interactions. Rigid bond constraints between hydrogen and any other atoms were applied through SHAKE / RATTLE algorithm. The integration time step was set as 2 fs with short-range nonbonded forces updated every 2 fs and long-range electrostatic forces updated every 4fs.

[0167] Initial configuration preparation and equilibration: To ensure that total number of ions and water molecules are correctly determined for all salts and concentrations, we first decided the simulation box sizes and thus ion numbers. All the simulation boxes were fixed to the size with three dimensions around 50 Å. Water molecules were initially placed using tLEaP program (Case, D. A. et al. The Amber biomolecular simulation programs. Journal of Computational Chemistry 26, 1668–1688 (2005)), the numbers of which were then adjusted according to volume changes after 100 ps minimization, 200 ps heating, and 2 ns constant pressure (NPT) equilibration. After the water molecule number corrections, another round of 100 ps minimization, 200 ps heating, 2 ns constant pressure (NPT) equilibration, plus 2 ns constant volume (NVT) equilibration, were conducted to ensure the correct setup and complete equilibration of simulation systems.

[0168] Water structure characterization: The equilibrated initial configurations containing water molecules and ions were prepared as stated above. The production simulations for water structure characterization were run for 100 ns with trajectories 29 ME1\53235995.v1DOCKET NO.117823-37020 recorded every 10 ps. The radial distribution function g(r) between cations (or anions) and oxygen atoms of water can be used to characterize local water structure around ions. It was calculated via VMD and averaged among all cations (or anions) and all trajectory frames with the definition aswhere ^^^^^^is the number of oxygen atoms of water within the shell of thickness at the distance r, 4πr2∙dr is the volume of the shell, ^^bulkis the bulk average density of oxygen atoms of water in the simulation box.

[0169] Water entropy calculation: The equilibrated initial configurations containing water molecules and ions were prepared as stated above. The production simulations for water entropy calculation were run for 20 ps (Lin, S.-T., Maiti, P. K. & Goddard, W. A. I. Two-Phase Thermodynamic Model for Efficient and Accurate Absolute Entropy of Water from Molecular Dynamics Simulations. J. Phys. Chem. B 114, 8191–8198 (2010)) with trajectories recorded every 2 fs. All the water entropy calculations were conducted based on Two-Phase Thermodynamic (2PT) model (Lin, S.-T., Maiti, P. K. & Goddard, W. A. I. Two-Phase Thermodynamic Model for Efficient and Accurate Absolute Entropy of Water from Molecular Dynamics Simulations. J. Phys. Chem. B 114, 8191–8198 (2010), Lin, S.-T., Blanco, M. & Goddard, W. A., III. The two-phase model for calculating thermodynamic properties of liquids from molecular dynamics: Validation for the phase diagram of Lennard-Jones fluids. The Journal of Chemical Physics 119, 11792–11805 (2003)) using corresponding open source program (https: / / github.com / atlas-nano / 2PT).

[0170] Protein free energy landscape construction: A short alanine-based peptide with 20 amino acids (AAAAKAAAAKAAAAKAAAAK), which has been confirmed experimentally (Marqusee, S., Robbins, V. H. & Baldwin, R. L. Unusually stable helix formation in short alanine-based peptides. Proc Natl Acad Sci U S A 86, 5286–5290 (1989)) to be a stable alpha helix structure in pure water, was adopted as the model protein to study the difference of protein free energy landscapes under different concentrations of LiBr. The atomistic protein structure was obtained through ColabFold v1.5.5 (Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat Methods 19, 679–682 (2022)), with N-terminus acetylated and C-terminus amidated using PyMOL 2.5.5. The equilibrated initial configurations containing protein, water molecules and ions were prepared as stated above. Metadynamics simulations 30 ME1\53235995.v1DOCKET NO.117823-37020 were implemented for protein free energy landscape construction using Colvars module (Fiorin, G., Klein, M. L. & Hénin, J. Using collective variables to drive molecular dynamics simulations. Molecular Physics 111, 3345–3362 (2013)) on top of standard molecular dynamics simulations as stated above. Root mean square displacement (RMSD) from the backbone of native alpha helix protein structure was selected as the collective variable to guide the sampling of unfolded confirmations. After the first run of metadynamics simulation, two more protein confirmations with medium RMSD (6 Å) and large RMSD (12 Å) respectively, were chosen as two other start structures. In total, nine metadynamics simulations for each LiBr concentration, started from three different initial protein conformations, were conducted. Each simulation was run for around 2 µs with trajectory recorded every 100 ps. Convergence of metadynamics simulations was examined using block analysis via the open-source, community- developed PLUMED library (Bonomi, M. et al. Promoting transparency and reproducibility in enhanced molecular simulations. Nat Methods 16, 670–673 (2019)), version 2.8.3 (Tribello, G. A., Bonomi, M., Branduardi, D., Camilloni, C. & Bussi, G. PLUMED 2: New feathers for an old bird. Computer Physics Communications 185, 604–613 (2014)). Six converged simulations out of nine for each LiBr concentration were used for final protein free energy landscape construction.

[0171] Analytical model of entropy penalty decrease: The protein unfolding reaction is defined as F⇆U, where F is the folded state and U is the unfolded state. For folded proteins, since they can maintain stable structures in pure water solution (or very low concentration NaCl solution), the free energy change ΔGwater of the unfolding reaction in pure water should be positive, i.e., ΔGwater > 0.

[0172] According to the unfolding experiments presented in this work, LiBr has the strongest capability of unfolding (denaturing) proteins, followed by LiCl. NaBr cannot denature those tested proteins in this work. Therefore, the free energy changes of the unfolding reaction in different solution environments can be ranked as follows.

[0173] Δ^^LiBr < Δ^^LiCl < Δ^^NaBr < Δ^^water,

[0174] where ΔGLiBr and ΔGLiCl for some proteins in high concentration salt solutions are negative (i.e., the unfolding reaction is favored).

[0175] The difference between free energy changes of the unfolding reaction in salt solutions ΔGsalt and that in pure water ΔGwater can be written as follows. 31 ME1\53235995.v1DOCKET NO.117823-37020

[0176] where enthalpy change difference ΔΔH equals to 0 given the ITC data (Fig. 11A and Fig.11b) that no direct interaction between proteins and ions was observed.

[0177] Thus, we have ΔΔ^^ = −^^ΔΔ^^ < 0, where ΔΔS is the entropy penalty decreasedue to high concentration salts and should be positive.

[0178] Entropy penalty decrease caused by high concentration salts can be written as follows. ΔΔ^^ = Δ^^salt − Δ^^water > 0= Δ^^protein salt + Δ^^ solutionsalt − (Δ^^protein water + Δ^^wsoaltuetrion) , =Δ^^ solutionsalt −solution water

[0179] where protein conformational entropy changes ΔSproteinduring unfolding is independent of solutions and thus cancel with each other, ΔSsolutionis the solution entropy change.

[0180] The solution entropy ^^solutionis composed of two parts, i.e., molecular entropy and network entropy. Molecular entropy is composed of translational, rotational, and vibrational entropy of water molecules themselves. Network entropy comes from the collective behavior of water network, which considers different combinations water molecules can have to form a network. Therefore, the solution entropy change during protein unfolding can be expressed as follows. Δ^^solution = Δ^^translation + Δ^^rotation + Δ^^vibration + Δ^^network.

[0181] Consider a water network with ^^ water molecules (so-called free water molecules since they are not bound by either proteins or ions), the network entropy of each water molecule is

[0182] given one water molecule can form four hydrogen bonds with other water molecules in the network. ^^Bis the Boltzmann constant. If the water molecule is taken out from the water network and bound to proteins or ions, its network entropy is decreased to 0. Therefore, network entropy change during protein unfolding can be expressed as

[0183] The entropy penalty decrease originated from the shrinkage of intact water network size in high salt concentration systems can be expressed as 32 ME1\53235995.v1DOCKET NO.117823-37020

[0184] where ^^wtoatatelr is the total number of water molecules in pure water systems as all water molecules here are considered as free , both ^^sfraelteandare normallymuch larger than 1 (i.e., ≫ 1 ), ^^ =^^ free salt^^totalwateris the ratio of free water number in salt solution to that of pure water system.

[0185] Finally, we have, ΔΔ^^ = Δ^^ solutionsolution salt − Δ^^water= ΔΔ^^translation + ΔΔ^^rotation + ΔΔ^^vibration + Δ^^network, =ΔΔ^^translation + ΔΔ^^rotation + ΔΔ^^vibration − 4^^Bln ^^

[0186] where ΔΔ^^translation, ΔΔ^^rotation, and ΔΔ^^vibrationcan be expressed as follows,

[0187] where j denotes translational or rotational or vibrational components of solution entropy, thejof bound water molecules by proteins are assumed to be independent of their solution environments and thus cancel out each other. All the parameters in above equation (ΔΔ^^translation, ΔΔ^^rotation, ΔΔ^^vibration, and ^^) can be obtained through molecular dynamics simulation and Two-Phase Thermodynamic (2PT) calculation (Lin, S.-T., Maiti, P. K. & Goddard, W. A. I. Two-Phase Thermodynamic Model for Efficient and Accurate Absolute Entropy of Water from Molecular Dynamics Simulations. J. Phys. Chem. B 114, 8191–8198 (2010); Lin, S.-T., Blanco, M. & Goddard, W. A., III. The two-phase model for calculating thermodynamic properties of liquids from molecular dynamics: Validation for the phase diagram of Lennard-Jones fluids. The Journal of Chemical Physics 119, 11792–11805 (2003)).

[0188] Thermogravimetric analysis: TGA of the LiBr solution prior to the 1st cycle and the recycled LiBr solution after the 5thcycle were conducted with a Discovery TGA 550. Around 30 mg of solution was loaded onto a platinum HT sample pan, stabilized under nitrogen flow for 10 min before measurement. Experiments were performed by heating samples to 100 °C at a rate of 5 °C min−1, then to 600 °C at a rate of 10 °C min−1in an air flow of 90 ml min−1. The mass of the sample was continuously measured 33 ME1\53235995.v1DOCKET NO.117823-37020 while the evolving gas from the sample was analyzed simultaneously by TG-FTIR. Data collection and analysis were carried out with TRIOS v.5.2.2 software.

[0189] Sample preparation: All samples were regenerated using α-keratin gel unless otherwise noted. The reverse mold for injection molding was created by casting PDMS (Sylgard™ 184, Dow Corning) around a 3D printed logo. Keratin gel was injected into the mold and transferred into a water bath until solidified . Film casting and dip coating followed a similar process by casting a layer of gel on glass coverslip or coating targets, then transferred into a water bath. Fiber spinning was achieved by injecting the gel through a 21 gauge needle (BD PrecisionGlide™) at a constant rate of 0.1 ml min-1into a bath of 0.4 M NaH2PO4 (Sigma-Aldrich) solution to accelerate the phase transition process. 3D printing was carried out using a Cellink BIO X 3D printer. The keratin gel was extruded into a supporting bath of 25% w / v Pluronic F127 (BASF) under an absolute pressure of 50 kPa and through a 22 gauge needle (Nordson EFD Precision Tips) that was moving at a speed of 4 mm s−1. The Pluronic bath was then removed by cooling and rinsing with water.

[0190] Keratin samples for mechanical tests, Raman and polarized Raman spectra were prepared by film casting a layer of keratin gel onto glass coverslips with a thickness around 0.4 mm, then transferred into a water bath to solidify the gel. Oxidized samples were fabricated by further transferring into a solution of 1% H2O2 for 1 h to reconnect the disulfide bonds. Finally, samples were sectioned into 5.20 mm rectangles and stored in water prior to measurements.

[0191] The tensegrity structure for the shape memory demonstration was created by oxidizing keratin films in a curled permanent state to form columns as compression units. These units were then interconnected with nylon threads (Singer®) to serve as tension units. Samples were fixed in a distorted temporary state by desiccating for 90 min with addition of external stress.

[0192] Mechanical tests: Mechanical tests were performed with a CellScale biaxial tester with 2.5 N load cells. For cyclic stretching curves, samples were tested at a strain rate of 5% per second to a maximum of 50% for 10 cycles. For yield strain measurements, samples were tested at a strain rate of 5% until fracture. Sample thickness was measured with a Mitutoyo Absolute Digimatic digital caliper. The modulus of reduced and oxidized samples was calculated within the 0-50% strain region.

[0193] Examples 34 ME1\53235995.v1DOCKET NO.117823-37020

[0194] The following examples are set forth as being representative of the present disclosure. These examples are not to be construed as limiting the scope of the present disclosure as these and other equivalent embodiments will be apparent in view of the present disclosure, figures and accompanying claims.

[0195] Example 1: Universal denaturation capability of LiBr.

[0196] To systematically investigate solute effects on protein conformation changes, a matrix of three proteins (Dihydrofolate Reductase (DHFR), fibronectin and alpha- keratin (wool)) representing different levels of structural complexities (Fig.1A, Fig. 1B and Fig.1C) were selected, along with three different combinations of monovalent ions including LiBr, LiCl, and NaBr.

[0197] Markedly distinct solute effects were observed for different proteins and salts across various concentrations, as evidenced by protein aggregation propensity from turbidity measurement at 405 nm (Fig.2A, Fig.2B and Fig.2C).

[0198] As the smallest protein tested, DHFR shows significant aggregation under 1 M LiBr and 5 M LiCl, while stays well-dispersed whatever NaBr concentrations (Fig. 2A).

[0199] Since both salt-induced precipitation and denaturation can result in protein aggregation, Fourier-transform infrared spectroscopy (FTIR) was used to further distinguish the underlying mechanisms (Fig.3A and Fig.3C).

[0200] Starting from 2 M LiBr, a gradual loss of secondary structures of DHFR was observed as concentration increases (Fig.3A), such as α-helix (1650-1655 cm-1) and β-sheet / turns (1660-1690 cm-1), along with an increase in aggregated β-like random structures (1620-1630 cm-1). The secondary structure loss of DHFR in 2 M LiBr was further confirmed by conformational kinetics measurement using quantitative Raman spectroscopy, where less-stable β-sheet disappeared first, followed by α-helix, with the obvious accumulation of aggregated random structures (Fig.4).

[0201] DHFR unfolding in ~ 5 M LiCl was also validated by tryptophan fluorescence measurement (Fig.7).

[0202] To further explore whether solutes can control protein conformations rather than purely denaturing proteins, fibronectin, a more complex protein comprised of two covalently linked extensible arms formed by β-sheet domains was chosen (Fig. 1B). 35 ME1\53235995.v1DOCKET NO.117823-37020

[0203] With the increase of protein structural complexity, higher concentrations of LiBr and LiCl (7 M and 8 M, respectively) were required to induce obvious protein aggregations of fibronectin (Fig.2B), where secondary structure loss was also confirmed by FTIR spectra (Fig.3B).

[0204] Before protein denaturation, protein conformational changes were observed through dynamic light scattering (DLS) measurements of the hydrodynamic radius Rh (Fig.5), where fibronectin transitions from the globular state (Rh ~ 8 nm) to the extended state (Rh ~23 nm) at a lower concentration of LiBr around 2 M.

[0205] By cross-referencing turbidity, FTIR, and DLS experiments, it is possible to identify a sequential protein structural dynamic in which the tertiary structures extension is preceded by the secondary structure loss as the concentration of LiBr increases (Fig.8).

[0206] Keratin structural deformation in LiBr solution that was observed by Feughelman et al. (Feughelman, M., Haly, A. R. & Mason, P. Contraction of Keratin Fibres in Aqueous Lithium Bromide. Nature 196, 957–958 (1962)) was also tested. In contrast to DHFR and fibronectin, keratin possesses hierarchical structures ranging from α-helices at the nanometer scale to protofibrils and filaments at the micrometer scale. A turbidity map generated from 24-hour soak of wool in salt solutions showed that visible signal can only be observed at high concentrations of LiBr (Fig.2C).

[0207] FTIR spectra further indicated the complete denaturation of both α-keratin from wool and β-keratin from goose feathers, irrespective of their different native configurations (Fig.3C).

[0208] Finally, scanning electron microscope (SEM) images of wool before (Fig.6A) and after (Fig.6B) treatment with 8 M LiBr, LiCl, and NaBr also corroborate previous findings, where only concentrated LiBr achieves denaturation of keratin (Figs.9A- 9F).

[0209] Example 2: Sustainable regeneration of keratin empowered by entropy-driven protein denaturation.

[0210] The entropy-driven mechanism due to the absence of direct interactions between proteins and denaturants is demonstrated in Fig.10.

[0211] Traditional denaturation methods using urea or guanidine hydrochloride involve direct protein-denaturant interactions, which not only lead to difficulties in 36 ME1\53235995.v1DOCKET NO.117823-37020 protein separation and further applications but also result in plenty of waste and pollution (Fig.10A).

[0212] Due to the absence of direct protein-denaturant interactions, entropy-driven protein denaturation via LiBr enables the sustainable regeneration of protein waste, featured by closed-loop LiBr recycling, simple protein separation, and facile further manufacturing (Fig.10B).

[0213] Example 3: Indirect solute effects.

[0214] Thermodynamically, protein conformational change or denaturation can be regulated by either enthalpy or entropy. To elucidate the protein denaturation mechanisms of LiBr (solute), isothermal titration calorimetry (ITC) experiments were conducted to explicitly measure the enthalpic contribution from direct solute-protein interactions, with commonly used denaturants urea and guanidine hydrochloride (GdnHCl) as control.

[0215] Prominent heat releases have been detected upon injection of urea or GdnHCl into DHFR and fibronectin (Fig.11A and Fig.11B). The scales of enthalpy change, dependent on protein sizes and exposures of hydrophobic domains (~ -7 kCal mol-1for DHFR and ~ -14 kCal mol-1for fibronectin), match the formation of 1-2 or 3-4 hydrogen bonds per organic molecule, respectively. These results are highly consistent with previous studies, where enthalpy-driven mechanisms of protein denaturation were established that direct solute-protein interactions overcome free energy penalty and thus facilitate the unfolding of proteins.

[0216] Remarkably, no enthalpy change was detected when LiBr solution was injected into protein samples under identical protocols, which provides the direct experimental evidence suggesting that solutes here do not engage in immediate interactions with proteins, instead, they modulate protein conformations by subtly adjusting solvent entropy.

[0217] Example 4: Entropy-driven protein denaturation mechanism.

[0218] The entropy-driven denaturation of LiBr was tested through molecular dynamics simulations and in order to obtain experimentally inaccessible atomistic- level insights.

[0219] First, the agreement of the simulation systems with experiments (Skipper, N. T. & Neilson, G. W. X-ray and neutron diffraction studies on concentrated aqueous solutions of sodium nitrate and silver nitrate. J. Phys.: Condens. Matter 1, 4141 37 ME1\53235995.v1DOCKET NO.117823-37020 (1989)) through local water density around ions were confirmed (Fig.12A and Fig. 12B).

[0220] The cooperativity of ion hydration (Tielrooij, K. J., Garcia-Araez, N., Bonn, M. & Bakker, H. J. Cooperativity in Ion Hydration. Science 328, 1006–1009 (2010)) was also observed, where local water structures are determined by both ions and their counterions. Molecular water entropy, encompassing both translational and rotational entropy of water molecules, was calculated based on TIP3P water model using Two-Phase Thermodynamic (2PT) method, the combination of which have been validated as the most accurate for replicating experimental entropy values (Lin, S.-T., Maiti, P. K. & Goddard, W. A. I. Two-Phase Thermodynamic Model for Efficient and Accurate Absolute Entropy of Water from Molecular Dynamics Simulations. J. Phys. Chem. B 114, 8191–8198 (2010)).

[0221] In both LiBr and LiCl solutions, two distinct populations of water molecules are detectable (Fig.13, Fig.14A and Fig.14B).

[0222] As suggested by experimental evidence of local effects of Li+ions (Roget, S. A., Heck, T. R., Carter-Fenk, K. A. & Fayer, M. D. Ion / Water Network Structural Dynamics in Highly Concentrated Lithium Chloride and Lithium Bromide Solutions Probed with Ultrafast Infrared Spectroscopy. J. Phys. Chem. B 127, 4532–4543 (2023)), the subset characterized by lower average entropy is identified as ion- bound water, whereas the other, possessing higher entropy, is regarded as free water that constitutes the water network through hydrogen bonds (Fig.12A, Fig. 12B, and Fig.15A).

[0223] Besides, LiBr solution also demonstrates a higher proportion of ionbound water compared to LiCl solution (Fig.13, Fig.14A, and Fig.14B). This enhanced capacity of Li+ions to sequester water in LiBr could stem from the more effective separation between Li+and Br- ions, attributable to the lower charge density of Br- ions and the substantial size disparity between Li+and Br- ions.

[0224] In contrast, the NaBr solution exhibits a single population akin to the intact hydrogen bond network of pure water (Fig.13 and Fig.16), which can be explained by the long-range global effects of ions on water dynamics in the absence of localized high charge density (Tielrooij, K. J., Garcia-Araez, N., Bonn, M. & Bakker, H. J. Cooperativity in Ion Hydration. Science 328, 1006–1009 (2010)) (Fig.15B).

[0225] The prominent difference of water network sizes between LiBr, LiCl, NaBr, and pure water, along with their disparate capabilities for protein denaturation without 38 ME1\53235995.v1DOCKET NO.117823-37020 direct protein interactions (Fig.2A, Fig.2B, Fig.2C, Fig.11A, and Fig.11B), inspired the introduction of network entropy, which accounts for different combinations water molecules can have to form a network given each water molecule has the potential to establish four hydrogen bonds with others in the network. The network entropy decrease (i.e., penalty) of a water molecule bound by proteins during unfolding can then be expressed as in Formula (1),

[0226] Δ^^network = −^^Bln((^^free− 1)(^^free− 2)(^^free− 3)(^^free− 4)), (1) where ^^Bis the Boltzmann constant, ^^freeis the free water number (i.e., the size of water network).

[0227] With the shrinkage of water network in salt solutions compared to pure water, network entropy penalty decreases by as in Formula (2), ΔΔ^^networkis the ratio of free water number in salt solution to that of pure water system. The reduction in network entropy penalty diminishes the energy barrier of protein unfolding, irrespective of any enthalpic gain from direct protein-solute interactions. The total entropy penalty reduction in salt solutions can then be calculated as in Formula (3) and as described in Materials and Methods. ΔΔ^^ = ΔΔ^^translation + ΔΔ^^rotation + ΔΔ^^vibration − 4^^B ln ^^ . (3)

[0228] Notably, the numerical results (Fig.17 and Fig.18) show a precise quantitative concordance with our experimental observations. For example, 3.5 M LiBr and 5 M LiCl possess the same level of entropy penalty reduction and exhibit similar protein denaturation capability (Fig.3A and Fig.7), while 6 M NaBr exhibits lower entropy penalty reduction than 2 M LiBr and thus cannot denature DHFR (Fig. 2A).

[0229] Furthermore, the decrease in network entropy penalty is observed to be the predominant factor over that of molecular entropy (Fig.18), the oversight of which has led to considerable debate regarding the effects of solutes on water dynamics and protein unfolding.

[0230] Finally, protein free energy landscapes in different LiBr concentrations were constructed using enhanced sampling method, where a stable α-helix peptide of 20 amino acids were adopted as the model protein (Fig.19). The increase of LiBr concentrations not only shifts the free energy landscapes in favor of unfolded states 39 ME1\53235995.v1DOCKET NO.117823-37020 but also lowers the energy barrier for unfolding, which lend further confirmation of our experimental observations and entropy-driven denaturation mechanism.

[0231] In conclusion, solute-induced water network disruption brings about the reduction of entropy penalty when proteins grab water molecules from bulk water network during unfolding, and thus facilitates the unfolding process and leads to protein denaturation (Fig.20).

[0232] Example 5: Keratin extraction protocol.

[0233] The pretreatment of keratin-rich sources (Angora wool, Merino wool shirt, and goose down from the R. H. Lindsay Wool Company, Merino Tech, and Dream Solutions USA, respectively) involves washing with ethanol (VWR 200 proof) three times to remove organic residues, followed by rinsing with water and allowing the material to dry at room temperature overnight. Subsequently, long wool fibers were cut into smaller fragments (less than 1 cm in length) to facilitate dispersion in solution.

[0234] During the extraction process, 10 g of keratin fragments were suspended in a 150 ml aqueous solution containing 8 M LiBr and 0.1 M DTT. The suspension was stirred for 36 h in an insulated environment at 90°C. Afterwards, insoluble residues were filtered through cotton cloth with 80 / 180 µm mesh size while still hot, then stored at 4°C overnight. A viscous, gel phase of aggregated keratin was observed, and centrifugation (3,000 r.p.m., 4°C) further separated the mixture into an upper phase of LiBr solution and a lower phase of keratin gel. The LiBr solution could then be easily collected and reused in the following extraction cycle along with a new batch of keratin source and DTT. A small amount of 8 M LiBr stock solution were added to compensate the solution loss from filtration process in order to maintain the total volume at 150 ml.

[0235] The keratin content of the aggregated gel was measured by taking 1 ml of gel and allowing it to fully solidify in water, freeze-dried, then lyophilized to obtain a dehydrated solid and measure its weight. It was found that 1 ml of keratin gel contains 392.8 ± 18.6 mg of α-keratin or 347.9 ± 23.7 mg of β-keratin (n = 6).

[0236] To calculate the extraction yield, the volume of the keratin gel obtained from each batch was measured, multiplied by the corresponding content, and divided by the initial weight of keratin fragments. The denatured keratin gel could be stably stored at 4°C in the absence of oxygen, with a shelf life longer than 6 months.

[0237] Example 6: Sustainable keratin regeneration with facile manufacturing. 40 ME1\53235995.v1DOCKET NO.117823-37020

[0238] Comparing with conventionally applied enthalpy-driven denaturants (e.g., urea, GdnHCl), the entropy-driven denaturation mechanism can address several major problems limiting the wide-application of protein regeneration, including the waste and pollution from single-used organic chemicals and the complexity of fabrication.

[0239] The invention described herein relates to the development of a sustainable and an economic protocol for keratin-rich waste regeneration with closed-loop recycling of LiBr solution followed by facile manufacturing (Fig.21).

[0240] In general, protein resources such as animal fragments or wool-based textiles are denatured by an aqueous solution of 8 M LiBr, with heat applied to accelerate reaction kinetics and prevent aggregation during extraction. A small amount of 1,4- dithiothreitol (DTT) is added as reductant to break down the dense disulfide matrix in keratin. After extraction, the mixture is hot filtered, cooled down and separated into a subnatant of denatured keratin gel and a supernatant of LiBr solution driven by spontaneous aggregation (Fig.22A, Fig.22B, and Fig.22C). Since the LiBr do not achieve denaturation via direct interactions, the resulting solution can be continuously recycled in a closed loop, which not only eliminates pollution from waste organics but also significantly reduces the consumption and cost of denaturants.

[0241] The cyclic stability of the closed-loop recycling protocol was validated by performing multiple cycles of extraction using both α-keratin from wool and β-keratin from goose feathers. The yield remains consistently around 40% after five cycles of extraction (Fig.23), while the first cycle exhibits a slightly lower yield possibly potentially due to a small amount of keratin remaining in the solution after separation. Unchanged composition of the recycled LiBr solution is further confirmed with thermogravimetric analysis (TGA) and coupled Fourier transform infrared spectroscopy (TG-FTIR) of the evolving gas (Fig.24, Fig.25A, and Fig. 25B).

[0242] Notably, in the absence of direct binding with denaturants, the spontaneous aggregation of denatured keratin enables a simple separation of condensed gel from the solution (Fig.26A), addressing difficulties with conventional organic reagents, such as urea (Fig.26B), which usually involves dialysis and lyophilization. Additionally, the separated keratin can undergo a fast phase transition process from denatured gel state to renatured solid state when transferring back into water (Fig. 41 ME1\53235995.v1DOCKET NO.117823-37020 26C), while keratin extracted with organic denaturants remains non-processable until fully dialyzed.

[0243] The viscous property of the gel and the fast phase transition process enable a wide array of manufacturing modalities including injection molding, film casting, dip coating, fiber spinning, and 3D printing (Fig.27) without any additional usage of organic solvents.

[0244] Example 7: Mechanical tunability of regenerated keratin.

[0245] Keratins, particularly α-keratins, process high content of cysteine residues, which form a dense network of intermolecular disulfide bonds. After extraction, these disulfide bonds are reduced to thiol groups, thereby endowing the regenerated keratin material with adjustable covalent crosslinking conditions through oxidation.

[0246] For instance, low level of crosslinking allows the α-helices to rearrange more freely under stress (Fig.28A and Fig.28C). As a result, reduced keratin preserves a high flexibility with a modulus of 124.3 ± 18.8 kPa and a yield strain of 435 ± 61%.

[0247] On the contrary, reconnecting the disulfide bonds can significantly increase the modulus of oxidized keratin by an order of magnitude to 1.35 ± 0.11 MPa and decrease the yield strain to 95 ± 9% (Fig.29, Fig.30, Fig.31A, Fig.31B, Fig.32A and Fig.32B). As shown in Fig.28A, Fig.28B, Fig.29, and Fig.30, tunable mechanical properties can be obtained by adjusting disulfide bond crosslinking in keratin.

[0248] Raman spectroscopy of keratin samples under 50% strain suggests that the majority of secondary structures remain unchanged within reduced keratin upon stretching, whereas a more discernible loss of α-helix domains and an increase of β / random structures can be observed in oxidized keratin (Fig.33A and Fig.33B). Differences between the two conditions reveal the effect of disulfide crosslinking to the α-helix network of regenerated keratin, providing the material with tunable mechanical properties from ductile to elastic (Fig.34).

[0249] In addition to enhancing the mechanical strength, disulfide crosslinking can also provide the keratin material with a hydration-responsive shape-memory property. Prior studies of Miserez et al. (Miserez, A., Wasko, S. S., Carpenter, C. F. & Waite, J. H. Non-entropic and reversible long-range deformation of an encapsulating bioelastomer. Nat Mater 8, 910–916 (2009)) and Cera et al. (Cera, L. et al. A bioinspired and hierarchically structured shape-memory material. Nat Mater 20, 242–249 (2021)) revealed that uniaxial strain can induce reversible uncoiling of 42 ME1\53235995.v1DOCKET NO.117823-37020 α-helix into metastable β-like structures within anisotropic bioelastomer. Similarly, in the crosslinked keratin network, the transition between coiled helix and uncoiled strands was also observed (Fig.28B), leading to a shape-memory effect facilitated by the locking and unlocking of hydrogen bonds. In a hydrated environment, dynamic reformation of hydrogen bonds enables the uncoiling of helices under external stress, allowing the material to be deformed into a temporary shape. Upon dehydration, the formation of intermolecular hydrogen bonds then locks the uncoiled chains, thereby fixing the network in the temporary shape. Finally, upon rehydration, presence of free water molecules can unlock the hydrogen bonds again, facilitating the recovery of native α-helix structures and returning the material to its permanent shape (Fig.35).

[0250] Example 8: Shape-memory capability of regenerated keratin.

[0251] The shape-memory capability of regenerated keratin is demonstrated both as a standalone object and as components of a more complex tensegrity structure (Fig. 36). Furthermore, the tissue engineering capability of keratin can be confirmed by neonatal rat ventricular myocytes (NRVMs) culturing, where the presence of keratin promotes cardiomyocyte monolayer formation and beating (Figs.37A to Fig.37C).

[0252] The sustainable re-generated keratin material of the invention, owing to its biocompatible nature, tunable mechanical properties, and shape-memory capability, holds promise for applications such as smart textiles or next-generation biomedical implants. 43 ME1\53235995.v1

Claims

DOCKET NO.117823-37020 CLAIMS What is claimed is:

1. A method for preparing regenerated keratin comprising: (a) providing a keratin-rich source; (b) contacting the keratin-rich source with an aqueous solution comprising about 6 M to about 12 M of a denaturing agent and about 0.05 M to about 0.5 M of a reducing agent to obtain a mixture; (c) heating the mixture from room temperature to about 90 °C for about 6 hours to about 72 hours to obtain an extracted mixture; (d) subjecting the extracted mixture to hot filtration to obtain a keratin solution; (e) cooling the keratin solution from about -20 °C to about -4 °C overnight to obtain a cooled keratin solution; (f) centrifuging the cooled keratin solution to obtain a subnatant comprising a denatured keratin gel and a supernatant comprising a solution of the denaturing agent; (g) reconstituting the subnatant comprising the denatured keratin gel in water to yield regenerated keratin; and (h) recycling the supernatant comprising a solution of the denaturing agent for use in steps (a) to (e), thereby preparing the regenerated keratin.

2. The method of claim 1, wherein the keratin-rich source is selected from animal fragments, feather, hair, skin, fingernail, hoof, wool-based textile, or a combination thereof.

3. The method of claim 2, wherein the feather is goose feather.

4. The method of claim 2, wherein the hair is animal hair or human hair.

5. The method of claim 4, wherein the hair is wool. 44 ME1\53235995.v1DOCKET NO.117823-37020 6. The method of any one of claims 1-5, wherein the keratin-rich source comprises of alpha-keratin, beta-keratin, or a combination thereof.

7. The method of any one of claims 1-5, wherein the denaturing agent is Lithum Bromide (LiBr).

8. The method of any one of claims 1-6, wherein the keratin-rich source is contacted with a solution comprising about 6.25 M, about 6.5, about 6.75 M, about 7 M, about 7.25 M, about 7.5, about 7.75 M, about 8 M, about 8.25 M, about 8.5, about 8.75 M, about 8 M, about 8.25 M, about 8.5, about 8.75 M, about 9 M, about 9.25 M, about 9.5, about 9.75 M, about 10 M, about 10.25 M, about 1.5, about 10.75 M, about 11 M, about 11.25 M, about 11.5, or about 11.75 M LiBr.

9. The method of claim 7, wherein the keratin-rich source is contacted with a solution comprising about 8 M LiBr.

10. The method of any one of claims 1-8, wherein the reducing agent is 1, 4- dithiothreitol (DTT).

11. The method of any one of claims 1-9, wherein the keratin-rich source is contacted with a solution comprising about 0.05 M, about 0.1 M, about 0.15 M, about 0.2 M, about 0.25 M, about 0.3 M, about 0.35 M, about 0.4 M, about 0.45 M, or about 0.5 M DTT.

12. The method of claim 10, wherein the keratin-rich source is contacted with an aqueous solution comprising about 0.1 M DTT.

13. The method of claim 1, wherein the said recycling in step (h) is a closed-loop recycling.

14. The method of claim 1, wherein the regenerated keratin is alpha-keratin.

15. The method of any one of claims 1-13, wherein the regenerated keratin is biocompatible.

16. The method of any one of claims 1-13, wherein the regenerated keratin is biodegradable. 45 ME1\53235995.v1DOCKET NO.117823-37020 17. The method of any one of claims 1-13, wherein the regenerated keratin has a tunable mechanical property.

18. The method of any one of claims 1-13, wherein the regenerated keratin has a shape-memory property.

19. A method of making a shape-memory object or structure, the method comprising: providing the regenerated keratin prepared according to the method of any one of claims 1-18; and forming a shape-memory object or structure from a material comprising the regenerated keratin via facile manufacturing.

20. The method of claim 19, wherein facile manufacturing comprises injection molding, film casting, film coating, dip coating, fiber spinning, fiber coating, or 3D printing.

21. The method of claim 20, wherein the shape-memory object or structure comprises smart textiles, biomedical implants, 3D-printed keratin sheets, yarns, threads, polymeric fibers, or fabrics. 46 ME1\53235995.v1

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