Adrien Lerbret

Evidence of a two-stage thermal denaturation process in lysozyme: A Raman scattering and differential scanning calorimetry investigation

Raman spectroscopy (in the low-frequency range and the amide I band region) and modulated differential scanning calorimetry investigations have been used to analyze temperature-induced structural changes in lysozyme dissolved in 1H2O and 2H2O in the thermal denaturation process. Low-frequency Raman data reveal a change in tertiary structure without concomitant unfolding of the secondary structure. Calorimetric data show that this structural change is responsible for the configurational entropy change associated with the strong-to-fragile liquid transition and correspond to about 1/3 of the native-denaturated transition enthalpy. This is the first stage of the thermal denaturation which is a precursor of the secondary structure change and is determined to be strongly dependent on the stability of the hydrogen-bond network in water. Low-frequency Raman spectroscopy provides information on the flexibility of the tertiary structure (in the native state and the transient folding state) in relation to the fragility of the mixture. The unfolding of the secondary structure appears as a consequence of the change in the tertiary structure and independent of the solvent. Protein conformational stability is directly dependent on the stability of the native tertiary structure. The structural transformation of tertiary structure can be detected through the enhanced 1H/2H exchange inhibited in native proteins. Taking into account similar features reported in the literature observed for different proteins it can be considered that the two-stage transformation observed in lysozyme dissolved in water is a general mechanism for the thermal denaturation of proteins.

Thermostabilization Mechanism of Bovine Serum Albumin by Trehalose

Thermal denaturation of bovine serum albumin (BSA) is analyzed from differential scanning calorimetry (DSC) and Raman spectroscopy investigations. DSC curves exhibit a marked dependence on protein concentration. BSA thermal denaturation becomes broader and bimodal, and the temperature of denaturation increases with increasing protein concentration. Raman scattering investigations simultaneously carried out in the low-frequency range (10-350 cm(-1)) and in the amide I band region (1500-1800 cm(-1)) indicate that the denaturation process is described as a biphasic process independent of protein concentration. The dependence of the protein stability upon the protein concentration can be interpreted from the coupling of protein and solvent dynamics. The confrontation of previous results obtained from Raman investigations on lysozyme (LYS) and the present study of BSA brings out significant information on protein dynamics and the coupling of protein and hydration-water dynamics in relation with the solvent accessible surface area. Contrary to LYS, the modification of the dynamics of hydration water by the protein is clearly observed on BSA. The influence of trehalose on the protein dynamics was analyzed. We found that trehalose reduces the dynamic fluctuations of polar side chains at the protein-solvent interface. The mechanism of thermostabilization by trehalose is related to the reduction of the exposure of hydrophobic groups of BSA to the water molecules, and to a strengthening of intermolecular O-H interactions in the hydrogen-bond network of water, leading to the stabilization of the tertiary structure.

Thermal Denaturation of Beta-Lactoglobulin and Stabilization Mechanism by Trehalose Analyzed from Raman Spectroscopy Investigations

The thermal denaturation process of beta-lactoglobulin has been analyzed in the 20-100 degrees C temperature range by Raman spectroscopy experiments simultaneously performed in the region of amide modes (800-1800 cm(-1)) and in the low-frequency range (10-350 cm(-1)). The analysis of amide modes reveals a two-step thermal denaturation process in the investigated temperature range. The first step corresponds to the dissociation of dimers associated with an increase of flexibility of the tertiary structure. In the second step, large conformational changes are detected in the secondary structure and described as a loss of alpha-helix structures and a concomitant formation of beta-sheets. Raman investigations in the low-frequency range provide important information on the origin of the denaturation process through the analysis of the solvent dynamics and its coupling with that of the protein. The softening of the tetrahedral structure of water induces the dissociation of dimers and makes the tertiary structure softer, leading to the water penetration in the protein interior. The methodology based on Raman investigations of amide modes and in the low-frequency region was used to analyze the mechanism of beta-lactoglobulin thermostabilization by trehalose. The main effect of trehalose is determined to be related to its capabilities to distort the tetrahedral organization of water molecules.

Molecular dynamics and neutron scattering study of glucose solutions confined in MCM-41.

Glucose aqueous solutions confined in MCM-41 cylindrical pores of diameter 3.2 nm have been studied by molecular dynamics (MD) simulations and quasielastic neutron scattering (QENS). MD simulations reveal a strong preferential interaction of glucose molecules with the silica walls, which induces significant concentration gradients within the pore. The influence of glucose on the structural and dynamical properties of water strongly depends on the region of the pore considered. The distortion of the hydrogen bond network (HBN) and of the tetrahedral organization of interfacial water molecules induced by silica is much stronger than that induced by glucose molecules. The interfacial glucose molecules diffuse about 1 order of magnitude slower than those in the core region. Differences in affinities for silica of the different species in confined hydrogen-bonded mixtures induce significant structural and dynamical heterogeneities not present in bulk solutions.

How Strongly Does Trehalose Interact with Lysozyme in the Solid State? Insights from Molecular Dynamics Simulation and Inelastic Neutron Scattering

Therapeutic proteins are usually conserved in glassy matrixes composed of stabilizing excipients and a small amount of water, which both control their long-term stability, and thus their potential use in medical treatments. To shed some light on the protein-matrix interactions in such systems, we performed molecular dynamics (MD) simulations on matrixes of (i) the model globular protein lysozyme (L), (ii) the well-known bioprotectant trehalose (T), and (iii) the 1:1 (in weight) lysozyme/trehalose mixture (LT), at hydration levels h of 0.0, 0.075, and 0.15 (in g of water/g of protein or sugar). We also supplemented these simulations with complementary inelastic neutron scattering (INS) experiments on the L, T, and LT lyophilized (freeze-dried) samples. The densities and free volume distributions indicate that trehalose improves the molecular packing of the LT glass with respect to the L one. Accordingly, the low-frequency vibrational densities of states (VDOS) and the mean square displacements (MSDs) of lysozyme reveal that it is less flexible-and thus less likely to unfold-in the presence of trehalose. Furthermore, at low contents (h = 0.075), water systematically stiffens the vibrational motions of lysozyme and trehalose, whereas it increases their MSDs on the nanosecond (ns) time scale. This stems from the hydrogen bonds (HBs) that lysozyme and trehalose form with water, which, interestingly, are stronger than the ones they form with each other but which, nonetheless, relax faster on the ns time scale, given the larger mobility of water. Moreover, lysozyme interacts preferentially with water in the hydrated LT mixtures, and trehalose appears to slow down significantly the relaxation of lysozyme-water HBs. Overall, our results suggest that the stabilizing efficiency of trehalose arises from its ability to (i) increase the number of HBs formed by proteins in the dry state and (ii) make the HBs formed by water with proteins stable on long (>ns) time scales.

Molecular dynamics studies of the conformation of sorbitol.

Molecular dynamics simulations of a 3 molal aqueous solution of D-sorbitol (also called D-glucitol) have been performed at 300 K, as well as at two elevated temperatures to promote conformational transitions. In principle, sorbitol is more flexible than glucose since it does not contain a constraining ring. However, a conformational analysis revealed that the sorbitol chain remains extended in solution, in contrast to the bent conformation found experimentally in the crystalline form. While there are 243 staggered conformations of the backbone possible for this open-chain polyol, only a very limited number were found to be stable in the simulations. Although many conformers were briefly sampled, only eight were significantly populated in the simulation. The carbon backbones of all but two of these eight conformers were completely extended, unlike the bent crystal conformation. These extended conformers were stabilized by a quite persistent intramolecular hydrogen bond between the hydroxyl groups of carbon C-2 and C-4. The conformational populations were found to be in good agreement with the limited available NMR data except for the C-2-C-3 torsion (spanned by the O-2-O-4 hydrogen bond), where the NMR data support a more bent structure.

Binding of Divalent Cations to Polygalacturonate: A Mechanism Driven by the Hydration Water.

We have investigated the interactions between polygalacturonate (polyGal) and four divalent cations (M(2+) = Ba(2+), Ca(2+), Mg(2+), Zn(2+)) that differ in size and affinity for water. Our results evidence that M(2+)-polyGal interactions are intimately linked to the affinity of M(2+) for water. Mg(2+) interacts so strongly with water that it remains weakly bound to polyGal (polycondensation) by sharing water molecules from its first coordination shell with the carboxylate groups of polyGal. In contrast, the other cations form transient ionic pairs with polyGal by releasing preferentially one water molecule (for Zn(2+)) or two (for Ca(2+) and Ba(2+)), which corresponds to monodentate and bidentate binding modes with carboxylates, respectively. The mechanism for the binding of these three divalent cations to polyGal can be described by two steps: (i) monocomplexation and formation of point-like cross-links between polyGal chains (at low M(2+)/Gal molar ratios, R) and (ii) dimerization (at higher R). The threshold molar ratio, R*, between these two steps depends on the nature of divalent cations and is lower for calcium ions (R* < 0.1) than for zinc and barium ions (R* > 0.3). This difference may be explained by the intermediate affinity of Ca(2+) for water with respect to those of Zn(2+) and Ba(2+), which may induce the formation of cross-links of intermediate flexibility. By comparison, the lower and higher flexibilities of the cross-links formed by Zn(2+) and Ba(2+), respectively, may shift the formation of dimers to higher molar ratios (R*).

Influence of pressure on the low-frequency vibrational modes of lysozyme and water: A complementary inelastic neutron scattering and molecular dynamics simulation study

We performed complementary inelastic neutron scattering (INS) experiments and molecular dynamics (MD) simulations to study the influence of pressure on the low‐frequency vibrational modes of lysozyme in aqueous solution in the 1 atm–6 kbar range. Increasing pressure induces a high‐frequency shift of the low‐frequency part (<10 meV = 80 cm−1) of the vibrational density of states (VDOS), g(ω), of both lysozyme and water that reveals a stiffening of the interactions ascribed to the reduction of the protein and water volumes. Accordingly, high pressures increase the curvature of the free energy profiles of the protein quasiharmonic vibrational modes. Furthermore, the nonlinear influence of pressure on the g(ω) of lysozyme indicates a change of protein dynamics that reflects the nonlinear pressure dependence of the protein compressibility. An analogous dynamical change is observed for water and stems from the distortion of its tetrahedral structure under pressure. Moreover, our study reveals that the structural, dynamical, and vibrational properties of the hydration water of lysozyme are less sensitive to pressure than those of bulk water, thereby evidencing the strong influence of the protein surface on hydration water. Proteins 2013.

Glucose interactions with a model peptide

Molecular dynamics simulations have been conducted of the helical polypeptide melittin, in concentrated aqueous solutions of the alpha and beta anomers of D‐glucopyranose. Glucose is an osmolyte, and it is expected to be preferentially excluded from the surfaces of proteins. This was indeed found to be the case in the simulations. The results indicate that the observed exclusion may have a contribution from an under‐representation of hydrogen bonding interactions between glucose groups and exposed side chains, compared to water. However, glucose was found to bind quite specifically to melittin by stacking its hydrophobic face, consisting of aliphatic protons, against the flat hydrophobic face of the indole group of the tryptophan‐19 side chain. Although the binding site for this interaction is localized, the binding is weak for both anomers, with a binding free energy estimated as only ∼0.5 kcal/mol (i.e. near kBT). The face of the sugar stacked against the Trp indole ring is different for the two anomers of glucose, due to the disruption of the H1‐H3‐H5 hydrophobic triad of the beta anomer by the axial C1 hydroxyl group in the alpha anomer. The measurable affinity of the sugar for the Trp side chain is consistent with the very frequent occurrence of this group in the binding sites of proteins that complex with sugars. Proteins 2011;

Molecular Packing, Hydrogen Bonding, and Fast Dynamics in Lysozyme/Trehalose/Glycerol and Trehalose/Glycerol Glasses at Low Hydration

Water and glycerol are well-known to facilitate the structural relaxation of amorphous protein matrices. However, several studies evidenced that they may also limit fast (∼picosecond-nanosecond, ps-ns) and small-amplitude (∼Å) motions of proteins, which govern their stability in freeze-dried sugar mixtures. To determine how they interact with proteins and sugars in glassy matrices and, thereby, modulate their fast dynamics, we performed molecular dynamics (MD) simulations of lysozyme/trehalose/glycerol (LTG) and trehalose/glycerol (TG) mixtures at low glycerol and water concentrations. Upon addition of glycerol and/or water, the glass transition temperature, Tg, of LTG and TG mixtures decreases, the molecular packing of glasses is improved, and the mean-square displacements (MSDs) of lysozyme and trehalose either decrease or increase, depending on the time scale and on the temperature considered. A detailed analysis of the hydrogen bonds (HBs) formed between species reveals that water and glycerol may antiplasticize the fast dynamics of lysozyme and trehalose by increasing the total number and/or the strength of the HBs they form in glassy matrices.