Hydration in Deep Eutectic Solvents Induces Non-monotonic Changes in the Conformation and Stability of Proteins

The preservation of labile biomolecules presents a major challenge in chemistry, and deep eutectic solvents (DESs) have emerged as suitable environments for this purpose. However, how the hydration of DESs impacts the behavior of proteins is often neglected. Here, we demonstrate that the amino acid environment and secondary structure of two proteins (bovine serum albumin and lysozyme) and an antibody (immunoglobulin G) in 1:2 choline chloride:glycerol and 1:2 choline chloride:urea follow a re-entrant behavior with solvent hydration. A dome-shaped transition is observed with a folded or partially folded structure at very low (40 wt % H2O) DES hydration, while protein unfolding increases between those regimes. Hydration also affects protein conformation and stability, as demonstrated for bovine serum albumin in hydrated 1:2 choline chloride:glycerol. In the neat DES, bovine serum albumin remains partially folded and unexpectedly undergoes unfolding and oligomerization at low water content. At intermediate hydration, the protein begins to refold and gradually retrieves the native monomer–dimer equilibrium. However, ca. 36 wt % H2O is required to recover the native folding fully. The half-denaturation temperature of the protein increases with decreasing hydration, but even the dilute DESs significantly enhance the thermal stability of bovine serum albumin. Also, protein unfolding can be reversed by rehydrating the sample to the high hydration regime, also recovering protein function. This correlation provides a new perspective to understanding protein behavior in hydrated DESs, where quantifying the DES hydration becomes imperative to identifying the folding and stability of proteinsA.S.F. acknowledges the Spanish Ministerio de Universidades for the awarded Maria Zambrano fellowship. Also, the research in this study was performed with financial support from Vinnova─Swedish Governmental Agency for Innovation Systems within the NextBioForm Competence Centre and from The Crafoord Foundation (grant #20190750). The authors thank the Institute Laue-Langevin for the awarded beamtime (8-03-1049)S

Structural diversity of native Major Ampullate, Minor Ampullate, Cylindriform, and Flagelliform silk proteins in solution

The foundations of silk spinning, the structure, storage, and activation of silk proteins, remain highly debated. By combining solution small-angle neutron and X-ray scattering (SANS and SAXS) alongside circular dichroism (CD), we reveal a shape anisotropy of the four principal native spider silk feedstocks from Nephila edulis. We show that these proteins behave in solution like elongated semiflexible polymers with locally rigid sections. We demonstrated that minor ampullate and cylindriform proteins adopt a monomeric conformation, while major ampullate and flagelliform proteins have a preference for dimerization. From an evolutionary perspective, we propose that such dimerization arose to help the processing of disordered silk proteins. Collectively, our results provide insights into the molecular-scale processing of silk, uncovering a degree of evolutionary convergence in protein structures and chemistry that supports the macroscale micellar/pseudo liquid crystalline spinning mechanisms proposed by the community

Structural changes in Nephila edulis silk proteins in solution

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Fabrication and Optimization of Stable, Optically Transparent, and Reusable pH-Responsive Silk Membranes

The fabrication of silk-based membranes that are stable, optically transparent and reusable is yet to be achieved. To address this bottleneck we have developed a method to produce transparent chromogenic silk patches that are optically responsive to pH. The patches were produced by blending regenerated silk fibroin (RSF), Laponite RD (nano clay) and the organic dyes neutral red and Thionine acetate. The Laponite RD played a central role in the patch mechanical integrity and prevention of dye leaching. The process was optimized using a factorial design to maximize the patch response to pH by UV absorbance and fluorescence emission. New patches of the optimized protocol, made from solutions containing 125 μM neutral red or 250 μM of Thionine and 15 mg/mL silk, were further tested for operational stability over several cycles of pH altering. Stability, performance, and reusability were achieved over the tested cycles. The approach could be extended to other reporting molecules or enzymes able to bind to Laponite

Dimerization of Terminal Domains in Spiders Silk Proteins Is Controlled by Electrostatic Anisotropy and Modulated by Hydrophobic Patches

The well-tuned spinning technology from spiders has attracted many researchers with the promise of producing high-performance, biocompatible, and yet biodegradable fibers. So far, the intricate chemistry and rheology of spinning have eluded us. A breakthrough was achieved recently, when the 3D structures of the N and C terminal domains of spider dragline silk were resolved and their pH-induced dimerization was revealed. To understand the terminal domains' dimerization mechanisms, we developed a protein model based on the experimental structures that reproduces charge and hydrophobic anisotropy of the complex protein surfaces. Monte Carlo simulations were used to study the thermodynamic dimerization of the N-terminal domain as a function of pH and ionic strength. We show that the hydrophobic and electrostatic anisotropies of the N-terminal domain cooperate constructively in the association process. The dipolar attractions at pH 6 lead to weakly bound dimers by forcing an antiparallel monomer orientation, stabilized by hydrophobic locking at close separations. Elevated salt concentrations reduce the thermodynamic dimerization constant due to screened electrostatic dipolar attraction. Moreover, the mutations on ionizable residues reveal a free energy of binding, proportional to the dipole moment of the mutants. It has previously been shown that dimers, formed at pH 6, completely dissociate at pH 7, which is thought to be due to altered protein charges. In contrast, our study indicates that the pH increase has no influence on the charge distribution of the N-terminal domain. Instead, the pH-induced dissociation is due to an adapted, loose conformation at pH 7, which significantly hampers both electrostatic and hydrophobic attractive interactions

Interaction of haemin with albumin-based macroporous cryogel : Adsorption isotherm and fluorescence quenching studies

Albumin-based cryogels for capturing haemin were synthesised by crosslinking different biomolecules, bovine serum albumin (BSA) and ovalbumin (OVA). The impact of the protein and coupling agent concentrations on cryogel’s mechanical properties, swelling ratios and polymerisation yields, as well as autoclaving as a post-treatment on the cryogel, were studied. We found that BSA (50 mg/ml) and the crosslinker (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, 46 mg/ml) formed a cryogel with optimum physical characteristics at a comparatively low protein concentration. The cryogel’s mechanical stability was increased using a double-layer cryogel approach by crosslinking the BSA proteins at subzero temperature inside an acrylamide and hydroxyethyl methacrylate premade cryogels. Batch binding and kinetic adsorption isotherms of haemin on the cryogels were assessed to evaluate their binding capacity toward the porphyrin molecule. The results showed that single-layer cryogels (BSA and OVA) had a higher capacity (∼0.68 mg/ml gel) and higher reaction rate constant towards haemin adsorption than double-layer gels. In contrast, the double-layer cryogels had higher mechanical strength than single-layer gels. The experimental results suggested that the cryogels followed the Freundlich model and the pseudo-second-order isotherm for batch adsorption and kinetics, respectively. The interaction between haemin and the gels was studied by fluorescence quenching. We found between 1.1 and 1.6 binding sites for different cryogels

Manganese oxide functionalized silk fibers for enzyme mimics application

The inorganic metal or metal-oxide nanoparticles (NPs) that mimic enzymes are of great interest due to improved physical and chemical properties compared with native enzymes. Here, we report that manganese dioxide (MnO2)-Silk exhibit catalase, oxidase, and peroxidase-like activities. The MnO2-Silk hybrid fibers effectively decomposed hydrogen peroxide (H2O2) and oxidized the typical horseradish peroxidase substrates, such as o-phenylenediamine (OPD), and 3,3′,5,5′- tetramethylbenzidine (TMB) in the presence or absence of H2O2. The oxidative properties of MnO2-Silk fiber hybrid showed an enzyme-like behavior for the catalase-like activity, oxidase-like activity, and peroxidase-like activity. The operational stability of the MnO2-Silk fiber hybrid over ten cycles showed a constant residual activity of about 25–30% after 2–3 cycles indicating that MnO2-Silk fiber hybrid could be used as a satisfactory oxidoreductase enzyme mimics. Potentiometric titration was used to determine the surface charges of the MnO2-Silk catalyst. Together, we identified the reactive species as Mn1−x4+Mnx3+O2−x(OH)x with a pK of approximately 5.2. Our results have implications on the understanding of the catalytic origin and interaction of metal oxides NP with various biomaterials

Sonication enhances the stability of MnO2 nanoparticles on silk film template for enzyme mimic application

We have developed an in-situ method using sonication (3 mm probe sonicator, 30 W, 20 kHz) and auto-reduction (control) to study the mechanism of the formation of manganese dioxide (MnO2) on a solid template (silk film), and its resulting enzymatic activity on tetramethylbenzidine (TMB) substrate. The fabrication of the silk film was first optimized for stability (no degradation) and optical transparency. A factorial approach was used to assess the effect of sonication time and the initial concentration of potassium permanganate (KMnO4). The result indicated a significant correlation with a fraction of KMnO4 consumed and MnO2 formation. Further, we found that the optimal process conditions to obtain a stable silk film with highly catalytic MnO2 nanoparticles (NPs) was 30 min of sonication in the presence of 0.5 mM of KMnO4 at a temperature of 20–24 °C. Under the optimal condition, we monitored in-situ the formation of MnO2 on the silk film, and after thorough rinsing, the in-situ catalysis of 0.8 mM of TMB substrate. For control, we used the auto-reduction of KMnO4 onto the silk film after about 16 h. The result from single-wavelength analysis confirmed the different kinetics rates for the formation of MnO2 via sonication and auto-reduction. The result from the multivariate component analysis indicated a three components route for sonication and auto-reduction to form MnO2-Silk. Overall, we found that the smaller size, more mono-dispersed, and deeper buried MnO2 NPs in silk film prepared by sonication, conferred a higher catalytic activity and stability to the hybrid material

Site-Specific Introduction of Negative Charges on the Protein Surface for Improving Global Functions of Recombinant Fetal Hemoglobin

Due to its compatible oxygen-transporting abilities, hemoglobin (Hb) is a protein of interest in the development of artificial oxygen therapeutics. Despite continuous formulation attempts, extracellular Hb solution often exhibits undesirable reactions when applied in vivo. Therefore, protein engineering is frequently used to examine alternative ways of controlling the unwanted reactions linked to cell-free Hb solutions. In this study, three mutants of human fetal hemoglobin (HbF) are evaluated; single mutants αA12D and αA19D, and a double mutant αA12D/A19D. These variants were obtained by site-directed mutagenesis and recombinant production in E. coli, and carry negative charges on the surface of the α-subunit at the designated mutation sites. Through characterization of the mutant proteins, we found that the substitutions affected the protein in several ways. As expected, the isoelectric points (pIs) were lowered, from 7.1 (wild-type) down to 6.6 (double mutant), which influenced the anion exchange chromatographic procedures by shifting conditions toward higher conductivity for protein elution. The biological and physiological properties of HbF could be improved by these small modifications on the protein surface. The DNA cleavage rate associated with native HbF could be reduced by 55%. In addition, the negatively charged HbF mutant had an extended circulation time when examined in a mouse model using top load Hb additions. At the same time, the mutations did not affect the overall structural integrity of the HbF molecule, as determined by small-angle X-ray scattering. In combination with circular dichroism and thermal stability, modest structural shifts imposed by the mutations could possibly be related to changes in secondary structure or reorganization. Such local deformations were too minor to be determined within the resolution of the structural data; and overall, unchanged oxidation and heme loss kinetics support the conclusion that the mutations did not adversely affect the basic structural properties of Hb. We confirm the value of adding negatively charged residues onto the surface of the protein to improve the global functions of recombinant Hb

Differential scanning fluorimetry illuminates silk feedstock stability and processability.

The ability to design and implement silk feedstock formulations for tailored spinning has so far eluded the bioengineers. Recently, the high throughput screening technique of differential scanning fluorimetry (DSF) demonstrated the link between the instability transition temperature (Ti) and the processability of the silk feedstock. Using DSF we screened a large set of chemicals known to affect solvent quality. A multivariate analysis of the results shows that, regardless of the diversity of chemicals, three groupings are significantly distinguishable: G1 = similar to native silk; G2 = largely dominated by electrostatic interactions; and G3 = dominated by chelating interactions. We propose a thermodynamic analysis based on a pre- and post-transition fit to estimate the van't Hoff enthalpies (ΔHv) and the instability temperature (Ti). Our analysis shows that the ΔTi and ΔHv values were distinct: G1 (ΔTi = 0.23 ± 0.2; ΔHv = -159.1 ± 5.6 kcal mol(-1)), G2 (ΔTi = -7.3 ± 0.7; ΔHv = -191.4 ± 5.5 kcal mol(-1)), and G3 (ΔTi = -19.9 ± 3.3; ΔHv = -68.8 ± 6.0 kcal mol(-1)). Our analysis further combined the ΔTi value and the ΔHv value using stability ΔΔG to find that G1 only marginally stabilizes native silks (ΔΔG = -0.15 ± 0.04 kcal mol(-1)), whereas G2 and G3 destabilize native silk (ΔΔG = 3.8 ± 0.11 and ΔΔG = 3.8 ± 0.3 kcal mol(-1), respectively). Here our analysis shows that native silk has a complex multistep transition that is possibly non-cooperative. However, all three groupings also show a direct and cooperative transition with varied stabilization effects. This analysis suggests that native silks are able to sample multiple substates prior to undergoing (or to delay) the final transition. We conclude by hypothesizing that the observed energetic plasticity may be mediated by a fragile packaging of the silk tertiary structure that is readily lost when the solvent quality changes