Stefania Cinelli

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.

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.

Structural Characterization of the pH-Denatured States of Ferricytochrome-c by Synchrotron Small Angle X-Ray Scattering

The ferricytochrome-c (cyt-c) shows a complex unfolding pathway characterized by a series of stable partially folded states. When titrated with HCl at low ionic strength, two transitions are detected. At pH 2, cyt-c assumes the U1 unfolded state, whereas the successive addition of Cl(-) ion from either HCl or NaCl induces the recompaction to a molten globule conformation (A1 and A2 states, respectively). A second unfolded state (U2) is also observed at pH 12. Recent data evidence different features for the local structure of the heme in the different states. To derive relationships between local and overall conformations, we analyzed the structural characteristics of the different states by synchrotron small angle X-ray scattering. The results show that in the acidic-unfolded U1 form the protein assumes a worm-like conformation, whereas in the alkaline-unfolded U2 state, the cyt-c is globular. Moreover, the molten globule states induced by adding HCl or NaCl to U1 appear structurally different: in the A1 state cyt-c is dimeric and less compact, whereas in the A2 form the protein reverts to a globular-like conformation. According to the local heme structure, a molecular model for the different forms is derived.

Effect of 1-alcohols on micelle formation and protein folding

Abstract The effect of monohydric alcohols on the micellization of sodium dodecyl sulphate (SDS) and on the thermal unfolding of lysozyme has been studied by DSC as a function of alcohol concentration in the water-rich region of composition. The results show that the effects due to alcohols on these very different systems and processes are strikingly similar and are closely linked with structural and dynamical properties of water–alcohol mixtures themselves. The data have been interpreted on the basis of the assumption that the addition of short chain alcohol affects both the thermal unfolding of proteins as the micellization process modifying the extent of enthalpy and entropy contribution associated with structural reorganization of water in these processes.

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.

pH-Dependent Local Structure of Ferricytochrome c Studied by X-Ray Absorption Spectroscopy

We have studied, using x-ray absorption spectroscopy by synchrotron radiation, the native state of the horse heart cytochrome c (N), the HCl denatured state (U(1) at pH 2), the NaOH denatured state (U(2) at pH 12), the intermediate HCl induced state (A(1) at pH 0.5), and the intermediate NaCl induced state (A(2) at pH 2). Although many results concerning the native and denatured states of this protein have been published, a site-specific structure analysis of the denatured and intermediate solvent induced states has never been attempted before. Model systems and myoglobin in different states of coordination are compared with cytochrome c spectra to have insight into the protein site structure in our experimental conditions. New features are evidenced by our results: 1) x-ray absorption near edge structure (XANES) of the HCl intermediate state (A(1)) presents typical structures of a pentacoordinate Fe(III) system, and 2) local site structures of the two intermediate states (A(1) and A(2)) are different.

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.

Dielectric Behavior of Lysozyme and Ferricytochrome-c in Water/Ethylene-Glycol Solutions

This work deals with a dielectric study at radio frequencies of the influence at room temperature of two organic molecules, known as cryo-protectants, ethylene-glycol and glycerol, on conformational and dynamic properties of two model proteins, lysozyme (lys) from chicken egg-white and ferricytochrome-c (cyt-c) from horse heart. Cyt-c is a compact globular protein whereas lys is composed of two structural domains, separated by the active site cleft. Measurements were carried out at the fixed temperature of 20 degrees C varying the concentration of the cosolvent up to 90% w/w. From the analysis of the dielectric relaxation of the protein solution, the effective hydrodynamic radius and the electric dipole moment of the protein were calculated as a function of the cosolvent concentration. The data show that glycerol does not modify significantly the conformation of both proteins and cyt-c is also stable in the presence of ethylene-glycol. On the contrary ethylene-glycol strongly affects the dielectric response of lysozyme denoting a specific effect on its conformation and dynamics. The data are coherently interpreted hypothesizing that glycol molecule wedges between and separates the two domains of lys making them rotationally independent.

Structural stability of ribosomes subjected to RNase treatment evidenced by dielectric spectroscopy and differential scanning microcalorimetry

Previous studies from our laboratory demonstrated the existence of at least two levels of structural complexity in E. coli 70S ribosomes. Ribosomal RNA seems to be principally involved in the overall stability of these structures. In this paper we present an investigation of ribosomes subjected to treatment with RNase. The study is based on both differential scanning microcalorimetry and dielectric spectroscopy. In the thermograms obtained on treated ribosomes only the low temperature peak of the two typical denaturation events observed in native ribosomes, is promptly eliminated by the enzyme treatment. Dielectric spectroscopy measurements carried out on the same samples indicate an alteration of the dielectric behavior previously shown to consist of two subsequent relaxation processes. In fact, only the low frequency relaxation is affected by the treatment. The second one, observed at higher frequency, remains unaltered. The same effect on the dielectric parameters is observed if the ribosome particles are heated and then cooled prior to measurement. These results are consistent with the idea that two different structures are present within the ribosome. One is very stable and withstands both temperature and RNase treatment while the second is promptly abolished by both treatments. Data presented here strongly suggest that the RNA domains exposed to the solvent play a fundamental role in the stability of the 3-D structure of the ribosome particle.