Preferential hydration fully controls the renaturation dynamics of collagen in water-glycerol solvents

Abstract

Glycerol is one of the additives which stabilize collagen, as well as globular proteins, against thermally induced denaturation -an effect explained by preferential hydration, i.e. by the formation, in water/glycerol solvents, of a hydration layer whose entropic cost favors the more compact triplehelix native structure against the denatured one, gelatin. Quenching gelatin solutions promotes renaturation which, however, remains incomplete, as the formation of a gel network gives rise to growing topological constraints. So, gelatin gels exhibit glass-like dynamical features such as slow aging of their shear modulus and stretched exponential stress relaxation, the study of which gives us access to the re(de)naturation dynamics of collagen. We show that this dynamics is independent of the bulk solvent viscosity and controlled by a single parameter, the undercooling ∆T below the glycerol-concentration dependent denaturation temperature. This provides direct proof of (i) the presence of a nanometer thick, glycerol-free hydration layer (ii) the high locality of the kinetically limiting process governing renaturation.

Direct Evidence for the Effect of Glycerol on Protein Hydration and Thermal Structural Transition

“…Combined experimental results and theoretical solutionscattering simulations yielded direct evidence on the effect of glycerol on the hydration (solvation) shell of myoglobin and the thermal structural transition. These results clearly indicate that the function of glycerol as a stabilizer of proteins is related to the preservation of protein hydration by the preferential exclusion of glycerol from the hydrationshell region of the protein at glycerol concentrations lower than $40% v/v, which agrees well with previous studies using different techniques (1)(2)(3)(4)(5)(6). At glycerol concentrations above $50% v/v, the partial penetration or replacement of glycerol into or with the hydration shell of water molecules surrounding the protein surface appears to depend on the glycerol concentration.…”

“…In the second model, glycerol molecules are preferentially excluded from the protein surface because of the hydration repulsion force. In this case, the hydration-shell density that is always higher than in bulk water is preserved and has a constant value even with increased glycerol (preferential exclusion model and preferential hydration model (1,6)). Although the above models are extreme cases induced by cosolutes depending on physicochemical conditions, it is meaningful to elucidate qualitatively typical effects of cosolutes on the scattering curve of protein.…”

“…A Raman and quasi-elastic inelastic neutron-scattering study by Caliskan et al showed that the rapid conformational fluctuations, low-frequency vibrations, and temperature variations of proteins are very sensitive to the behavior of solvents (5). The recent rheometer study by Ronsin et al on the effect of glycerol on the dynamics of collagen re(de)naturation suggested the presence of a nanometerthick, glycerol-free hydration layer where glycerol is completely expelled (6). Despite this collective data, there is little direct evidence showing the effect of glycerol on the protein structure, its preferential hydration (solvation) layer, and thermal stability.…”

Direct Evidence of Effect of Glycerol on Hydration and Helix-to-Sheet Transition of Myoglobin

“…By the combination of experimental results and theoretical solution scattering simulations, we have obtained a direct structural evidence on the effect of glycerol on the hydration-shell of myoglobin and the initial process of amyloid transition of myoglobin induced by temperature variation. The present results clearly indicate that the function of glycerol as a stabilizer of proteins is related to the preservation of protein hydration by the preferential exclusion of glycerol from the hydration-shell region of the protein at low glycerol-concentration (≤ ~40 % v/v), which agree well with the previous studies using different techniques [1][2][3][4][5][6]. The results at high glycerol-concentration (≥ ~50 % v/v), suggests the appearance of the preferential solvation (partial preferential penetration or replacement of glycerol into or with hydration-shell water surrounding the protein surface).…”

“…The Ramman and quasi-elastic inelastic neutron scattering study of the effect of glycerol on lysozyme showed that the protein's fast conformational fluctuations and low-frequency vibrations and its temperature variations are very sensitive to behavior of the solvents [5]. The recent rheometer study on the effect of glycerol on the dynamics of collagen re(de)naturation suggests the presence of a nanometer thick, glycerol-free hydration layer where glycerol is fully expelled out of it [6]. In spite of many previous studies, there are fewer direct evidence showing the effect of glycerol on the protein structure, its preferential hydration layer and thermal stability.…”