Entropy-driven denaturation enables sustainable protein regeneration through rapid gel-solid transition

Abstract

The upcycling of protein materials has long been hindered by the difficulty in restructuring them to usable forms. In contrast to proteins extracted using conventional organic denaturants, keratin treated with concentrated inorganic lithium bromide (LiBr) solution undergoes spontaneous aggregation into a stable gel with rapid phase-transition capability. We hypothesize that this distinct behaviour arises from an alternative denaturation mechanism that does not rely on direct interactions between proteins and concentrated ions. To investigate this, we study the denaturation effects of concentrated inorganic ion pairs using thermodynamic and spectroscopic analyses combined with atomistic molecular simulations. Through the isolation of indirect solute effects, our findings suggest a universal mechanism of salt-induced denaturation driven by entropy instead of enthalpy. We find that concentrated ion pairs like LiBr disrupt the water network structure rather than directly interacting with proteins. The mechanistic insight enables us to refine our previous extraction process of keratin materials, allowing for the spontaneous separation of denatured keratin into a condensed gel phase without additional chemicals and achieve closed-loop recycling of the LiBr denaturant. This simple, effective strategy can repurpose protein resources into versatile biomaterials in a simple, effective way without the need to separate organic denaturants from bulk proteins.

Fig. 1. Spontaneous aggregation of keratin gel with rapid phase transition capability.

a Extraction process of the room-temperature stable keratin gel using LiBr-induced denaturation. b Rheology measurements of the keratin gel, showing an increase in viscosity and shear-thinning property upon decrease of temperature. Data are presented as mean ± s.d. (n = 3 independently prepared keratin gel samples). c Keratin content of the aggregated gel after separation. Data are presented as mean ± s.d. (n = 6 independently prepared keratin gel samples). d Phase transition study illustrating the behavior of extracted keratin when immersed in water using coverslips with transparent well spacers. Keratin gel (i) obtained through LiBr extraction rapidly transitioned into a white solid phase, whereas keratin solution (ii) extracted with urea exhibited slow diffusion and remained soluble. Scale bar, 5 mm. e Facile manufacturing strategies including injection molding, film casting, dip coating, fiber spinning, and 3D printing. Scale bars, 1 cm (optical images), 300 µm (SEM images). Source data are provided as a Source Data file.

Fig. 2. Concentrated LiBr solutions induce universal protein conformational change and cause denaturation.

a Schematics of DHFR, fibronectin, and α-keratin (wool), representing three levels of structural complexity. b–d Turbidity assay of three respective proteins 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 = 4 independently prepared protein solutions). e–g FTIR of the amide I band indicate a gradual loss of secondary structure upon increasing of LiBr concentration. h Percentage change of the secondary structure in DHFR in 2 M LiBr, deconvoluted from the amide I band of Raman spectra. i DLS measurement of the hydrodynamic radius of fibronectin with increasing LiBr concentration, indicating an extension of quaternary structure followed by aggregation. Box plots represent the interquartile range (25th to 75th percentile), with the center line indicating the median. Whiskers extend to the minimum and maximum values. (n = 6 independently prepared protein solutions). j SEM image of wool prior (i) and after (ii) denaturation with 8 M LiBr. Scale bars, 50 µm. Source data are provided as a Source Data file.

Fig. 3. Protein conformational changes come from indirect solute effects manifested as water network entropy.

Reaction enthalpy of interactions between proteins (a, DHFR, b, fibronectin) and denaturants (LiBr, urea, and GdnHCl) as measured by isothermal titration calorimetry. Data are presented as mean ± s.d. (n = 3 independently prepared protein and denaturant solutions). c Distributions of molecular entropy of water molecules in 7 M LiBr, LiCl, and NaBr from MD simulations. d Total water entropy penalty decrease in different concentrations of LiBr, LiCl, and NaBr solutions. Data are obtained from MD simulations and calculated via the analytical model (Eq. 1). Data are presented as mean ± s.d. (n = 3 independent simulation runs), however, the error bars are smaller than the data point size. e Schematics of different ion effects on water structures. For LiBr and LiCl, high charge density of Li⁺ ion results in localized (trapped) water molecules and disrupted water network (i), while NaBr induces a more global effect without breaking water network (ii). f Contribution of water network entropy penalty decrease to the total water entropy penalty decrease. g Strong correlation between the theoretical value of water network entropy penalty decrease and the protein denaturation ratio observed from FTIR experiments in LiBr and LiCl solutions. The Pearson correlation coefficient is shown with a two-tailed P value of $3.44\times {10}^{-11}$. Dashed line represents the best fit. h Free energy landscape of an α-helix peptide (20 amino acids) in pure water and 1, 4, 7 M LiBr from MD simulations. Source data are provided as a Source Data file.

Fig. 4. Tunable mechanical properties and shape-memory effect of regenerated keratin.

a Schematic of the α-keratin in reduced state and directional rearrangement of α-helices induced by stretching. b Cyclic loading of reduced keratin to 50% strain. c Stress-strain curves of reduced and oxidized keratin until fracture. Scale bars, 2 mm. d Schematic of the α-keratin in oxidized state and uncoiling of helices upon stretch. e Cyclic loading of oxidized keratin to 50% strain. f Change of secondary structure percentage under 50% strain quantified from Raman deconvolution of amide I band. A more significant decrease of α-helix can be observed within oxidized sample, indicating the uncoiling of helices into metastable β-sheets. Data are presented as mean ± s.d. (n = 3 independently prepared keratin film samples). g Schematic of the shape memory effect triggered by hydration signal. h Demonstration of shape memory effect with a badge model and a tensegrity structure. Scale bars, 3 cm. Source data are provided as a Source Data file.

Fig. 5. Closed-loop recycling of LiBr solution.

a, Schematic showing the regeneration protocol of keratin waste with closed-loop recycling of LiBr solution. b Extraction yield of keratin extracted from wool and keratin extracted from goose feathers. Data are presented as mean ± s.d. (n = 7 for wool, n = 3 for feathers, independent extraction experiments). c Comparison of the TGA profile shows no major change in the LiBr solution after 5 cycles of extraction. d, e TG-FTIR of the LiBr solution recycled after keratin separation. f Environmental impact assessment of producing 1 kg keratin via agriculture (raw wool), urea extraction, and this work. Calculations conducted using the Intergovernmental Panel on Climate Change (IPCC) 100-year time horizon global warming potentials (GWP 100a) with ecoinvent 3.9.1 database. Detailed calculations described in Supplementary Tables 3 and 4. g Different keratin samples were regenerated from (i) wool, (ii) goose feathers, (iii) wool clothes, and (iv) human hair. Keratin samples were shaped into the word describing their sources using the injection molding method. Scale bars, 1 cm. Source data are provided as a Source Data file.