Unfolding and aggregation of lysozyme: a thermodynamic and kinetic study by FTIR spectroscopy.

The unfolding of hen egg-white lysozyme dissolved both in D(2)O and CH(3)CH(2)OD/D(2)O was studied by Fourier Transform Infrared (FTIR) absorption spectroscopy at different protein concentrations. A detailed description of the local and global rearrangement of the secondary structure upon a temperature increase, in the range 295 to 365K, was obtained through the analysis of the amide I band. Thermodynamic parameters for the melting, and the effect of the co-solvent in determining a change in thermal stability of the protein were evaluated. The protein-protein interactions were also followed as a function of temperature: a strong dependence of the cluster stability and aggregation yield on the solvent composition was observed. Finally, FTIR spectra taken at successive time steps of the aggregation enabled intermolecular contacts to be monitored as a function of time, and kinetic information to be obtained showing that both unfolded and folded states of lysozyme act as reactants for the clustering event.

Effect of head group size, temperature and counterion specificity on cationic micelles.

The critical micelle concentration (cmc) and ionisation degree (α), of micelles of cetyltrimethylammonium bromide (CTABr), cetyltrimethylammonium chloride (CTACl), cetyltripropylammonium bromide (CTPABr) and cetyltripropylammonium chloride (CTPACl) have been measured over a narrow temperature range at 2 degree intervals using electrical conductivity. CTPACl and CTPABr are very soluble in water and were measured in the temperature range 275.15-323.15K. The Krafft temperatures for CTABr and for CTACl are 293.15K and 284.15K, respectively and established a lower temperature limit for our studies on these two surfactants. The cmc vs temperature curves have a smooth minimum near room temperature and α linearly increases with temperature. The changes of cmc and α with temperature are smaller than those associated with the modification of head group size or counterion nature. Using these results, basic thermodynamic quantities associated with the phenomena of micellization have been evaluated. Thermodynamic properties of the surfactant solutions were discussed in terms of temperature dependence of the free energy, enthalpy and entropy of micellization. A close similarity between the effects of change in temperature on protein folding and micellization process appears from the data.

Preferential solvation of lysozyme in water/ethanol mixtures.

We provide a quantitative description of the solvation properties of lysozyme in water/ethanol mixtures, which has been obtained by a simultaneous analysis of small-angle neutron scattering and differential scanning calorimetry experiments. All data sets were analyzed by an original method, which integrates the exchange equilibrium model between water and ethanol molecules at the protein surface and activity coefficients data of water/ethanol binary mixtures. As a result, the preferential binding of ethanol molecules at the protein surface was obtained for both native and thermal unfolded protein states. Excess solvation numbers reveal a critical point at ethanol molar fraction ≈0.06, corresponding to the triggering of the hydrophobic clustering of alcohol molecules detected in water/ethanol binary mixtures.

Influence of glycerol on the structure and thermal stability of lysozyme: a dynamic light scattering and circular dichroism study.

Photon correlation spectroscopy and circular dichroism have been used to study the role of hydration in the structure and thermostability of the model protein lysozyme in water-glycerol mixtures. Two cases have been considered: water-rich and glycerol-rich regimes of concentrations. We follow the thermal denaturation both by optical spectral changes and hydrodynamic radius variations. This methodology allows us to emphasize the relevant role played by hydrophobic interactions during the process in aqueous solutions and, in glycerol, to distinguish the non-cooperative melting of secondary structure, supporting the view of a protein transition to a molten globule-like state.

Microcalorimetric study of thermal unfolding of lysozyme in water/glycerol mixtures: an analysis by solvent exchange model.

Folded protein stabilization or destabilization induced by cosolvent in mixed aqueous solutions has been studied by differential scanning microcalorimetry and related to difference in preferential solvation of native and denatured states. In particular, the thermal denaturation of a model system formed by lysozyme dissolved in water in the presence of the stabilizing cosolvent glycerol has been considered. Transition temperatures and enthalpies, heat capacity, and standard free energy changes have been determined when applying a two-state denaturation model to microcalorimetric data. Thermodynamic parameters show an unexpected, not linear, trend as a function of solvent composition; in particular, the lysozyme thermodynamic stability shows a maximum centered at water molar fraction of about 0.6. Using a thermodynamic hydration model based on the exchange equilibrium between glycerol and water molecules from the protein solvation layer to the bulk, the contribution of protein-solvent interactions to the unfolding free energy and the changes of this contribution with solvent composition have been derived. The preferential solvation data indicate that lysozyme unfolding involves an increase in the solvation surface, with a small reduction of the protein-preferential hydration. Moreover, the derived changes in the excess solvation numbers at denaturation show that only few solvent molecules are responsible for the variation of lysozyme stability in relation to the solvent composition.

Structure, stability, and activity of myoglobin adsorbed onto phosphate-grafted zirconia nanoparticles.

The adsorption of myoglobin (Mb) onto phosphate grafted-zirconia (ZrO2-P) nanoparticles was studied in terms of conformational studies and thermal stability, determined by circular dichroism (CD), differential scanning calorimetry (DSC), and atomic force microscopy (AFM). The changes in protein structure have been correlated with the catalytic activity of free and adsorbed Mb. CD and DSC studies indicate marked rearrangements in Mb structure upon adsorption onto phosphate-grafted zirconia nanoparticles. These structural rearrangements of Mb could be responsible for the loss of catalytic activity observed for the adsorbed Mb. In particular, the conformational changes due to the adsorption process induced a reduction of kcat and KM. AFM measurements indicate that the interaction with the grafted-zirconia nanoparticles also affects the morphology of the bound protein, inducing the nucleation of prefibrillar-like aggregates.

Preferential hydration of lysozyme in water/glycerol mixtures: a small-angle neutron scattering study.

In solution small-angle neutron scattering has been used to study the solvation properties of lysozyme dissolved in water/glycerol mixtures. To detect the characteristics of the protein-solvent interface, 35 different experimental conditions (i.e., protein concentration, water/glycerol fraction in the solvent, content of deuterated compounds) have been considered and a suitable software has been developed to fit simultaneously the whole set of scattering data. The average composition of the solvent in the close vicinity of the protein surface at each experimental condition has been derived. In all the investigated conditions, glycerol resulted especially excluded from the protein surface, confirming that lysozyme is preferentially hydrated. By considering a thermodynamic hydration model based on an equilibrium exchange between water and glycerol from the solvation layer to the bulk, the preferential binding coefficient and the excess solvation number have been estimated. Results were compared with data previously derived for ribonuclease A in the same mixed solvent: even if the investigated solvent compositions were very different, the agreement between data is noticeable, suggesting that a unique mechanism presides over the preferential hydration process. Moreover, the curve describing the excess solvation number as a function of the solvent composition shows the occurrence of a region of maximal hydration, which probably accounts for the changes in protein stability detected in the presence of cosolvents.

Some properties of lysozyme--lithium perfluorononanoate complexes.

Mixtures containing lysozyme, LYSO, and a fully fluorinated surfactant, lithium perfluorononanoate, LiPFN, were investigated in a wide range of concentrations and mole ratios. To ensure consistency to the data, a comparison was made, when possible, with the more conventional SDS as surfactant. Molecular solutions, precipitates, and micellar phases have been observed. The region of existence for each phase depends on the LiPFN/LYSO mole ratios, r, and was determined by different experimental methods. Optical absorbance, CD, 19F NMR, viscosity, electrical conductivity, and dielectric relaxation methods were used. Some methods give information on the protein conformation, others on the state of the surfactant or on the collective system properties, respectively. Addition of LiPFN gives rise to a solution, a poly phase dispersion (at low surfactant to protein ratios) and to a micelle-mediated redissolution of the precipitates. Concomitant to the above macroscopic properties, peculiar effects in the state of LYSO are observed. Low amounts of surfactant reduce significantly the amount of alpha-helix in favor of the beta-sheet conformation of the protein. The former is almost completely regained once micelle-assisted redissolution of the complex occurs. The tertiary structure of the protein, conversely, is lost at low surfactant content and never recovered. Such evidence suggests the occurrence of a molten globule conformation for LYSO in micellar media.

Modulation of hydrophobic effect by cosolutes.

This work concerns a comparison of the hydration properties and self-association behavior in aqueous solution of three biologically relevant simple molecules: tert-butyl alcohol (TBA), trimethylamine-n-oxide (TMAO), and glycine betaine (GB). These molecules were used as a model to study hydrophobic behavior in water solutions. In particular, water perturbation induced by TBA, TMAO, and GB molecules was studied as a function of the solute molar fraction X(2) (0 < X(2) < 0.04) by Raman spectra of water in the fundamental OH-stretching region (3,800-2,800 cm(-1)). Furthermore, possible hydrophobic clustering of these molecules was investigated by studying the behavior of the alkyl CH stretching band in the 3,100-2,900 cm(-1) frequency region as a function of X(2). To establish the existence of a correlation between the effects of these three solutes on the micellization process and changes in the properties of the solvent, the behavior of the critical micelle concentration of sodium dodecyl sulfate was also investigated as a function of the added amount of TBA, TMAO, and GB. On the whole, these data show that there is no direct correlation between a solute's effect on the water structure and its effect on micelle or protein stability. Results indicate that, while TBA starts to self-aggregate at approximately X(2) = 0.025, both TMAO and GB do not exhibit any significant self-aggregation up to the highest concentration considered. In addition, nonadditive perturbations of the H-bonded networks of solvent water are observed in the case of TBA solutions, but are absent in both the TMAO and GB cases. The absence of these nonlinear effects in TMAO and GB water solutions allow for tracing the microscopical mechanism of the neutrality of these osmolytes toward hydrophobic effects. This confers the compatibility to these two osmolytes, which can be accumulated at high concentrations without interfering with biochemical processes in the cell.

Picosecond-time-scale fluctuations of proteins in glassy matrices: the role of viscosity.

Through elastic neutron scattering we investigated the fast dynamics of lysozyme in hydrated powder form or embedded in glycerol-water and glucose-water matrices. We calculated the relaxational contribution to the mean square displacements of protein hydrogen atoms. We found that the inverse of this quantity is linearly proportional to the logarithm of the viscosity of the solvent glassy matrix. This relationship suggests a close connection between the picosecond-time-scale dynamics of protein side chains and the solvent structural relaxation.

Diffusive dynamics of water in tert-butyl alcohol/water mixtures.

Quasielastic neutron scattering has been used to investigate the dynamical behavior of H(2)O in water/tert-butyl alcohol solutions. The measurements were made at fixed temperature (293 K) as a function of tert-butyl alcohol molar fraction, x, in the range 0-0.042. The data have been compared to those of pure water in the temperature range 269-293 K. The effect of tert-butyl alcohol addition on water dynamics is equivalent to that obtained by lowering the temperature of pure water by an amount proportional to the alcohol concentration. The temperature dependence of the diffusivity parameters in pure water and their concentration dependence in tert-butyl alcohol/water solutions can be rescaled to a common curve attributing to each solution a concentration-dependent "structural temperature" lower than the actual thermodynamic one. These results can be understood in terms of Stillinger's picture of water structuring and of other more recent theoretical pictures that emphasize the influence of the geometrical properties of hydrogen bond networks on water mobility.

Hydration-dependent internal dynamics of reverse micelles: a quasielastic neutron scattering study.

We studied the overall atomic mobility of sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles in deuterated cyclohexane (C6D12) as a function of the molar ratio W=[D2O]/[AOT] with an incoherent quasielastic neutron scattering experiment at high energy resolution. For the almost anhydrous sample, the quasielastic broadening can be entirely attributed to the reverse micelle global motion, by considering explicitly both the rotational and the translational terms. As W increases above a threshold value W approximately 1 a wide quasielastic signal appears, which has been interpreted as the onset of a hydration-dependent intrinsic micelle dynamics. Such a contribution, which involves the AOT monomer hydrogen atoms, has a characteristic time of 0.2 ns. This result has been compared with previous dielectric measurements, which detected a relaxation process of the AOT fully hydrated head groups with the same characteristic time. The internal macromolecule mobility evaluated as a function of W numerically correlates with that of the mobile head groups, calculated by dielectric measurements. These findings suggest that both the hydrophobic and hydrophilic moieties dynamics is activated by the progressive hydration of the reverse micelle.

Effect of the environment on the protein dynamical transition: a neutron scattering study.

We performed an elastic neutron scattering investigation of the molecular dynamics of lysozyme solvated in glycerol, at different water contents h (grams of water/grams of lysozyme). The marked non-Gaussian behavior of the elastic intensity was studied in a wide experimental momentum transfer range, as a function of the temperature. The internal dynamics is well described in terms of the double-well jump model. At low temperature, the protein total mean square displacements exhibit an almost linear harmonic trend irrespective of the hydration level, whereas at the temperature T(d) a clear changeover toward an anharmonic regime marks a protein dynamical transition. The decrease of T(d) from approximately 238 K to approximately 195 K as a function of h is reminiscent of that found in the glass transition temperature of aqueous solutions of glycerol, thus suggesting that the protein internal dynamics as a whole is slave to the environment properties. Both T(d) and the total mean square displacements indicate that the protein flexibility strongly rises between 0.1 and 0.2h. This hydration-dependent dynamical activation, which is similar to that of hydrated lysozyme powders, is related to the specific interplay of the protein with the surrounding water and glycerol molecules.