G. Onori

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.

Self‐association of monohydric alcohols in water: Compressibility and infrared absorption measurements

The infrared absorption spectra of aqueous methanol, ethanol, 1‐ and 2‐propanol, t‐butanol, and n‐butoxyethanol in the region of asymmetric and symmetric C–H stretching modes (3200–2800 cm−1) have been measured at 25 °C as a function of alcohol concentration in the whole cosolvent mole fraction region. Measurements of adiabatic compressibility were also performed on the same water–alcohol solutions at 25 °C. The two sets of experimental data are compared and discussed in terms of a possible mechanism of molecular aggregation in the various regions of alcohol concentration.

Structural properties of water-ethanol mixtures: A correlation with the formation of micellar aggregates

Abstract The effect of ethanol on the critical micelle concentration (CMC) of several surfactant aqueous solutions has been studied by surface tension methods. The results show that with increasing alcohol concentration the CMC values first reach a minimum at an intermediate ethanol mole fraction x2* ≈ 0.055 and then increase with increasing x2. The value of x2* is close to that at which structural changes in the mixture occur as inferred from compressibility and optical absorption measurements. The present experiments support the assumption that the dominant mechanism by which ethanol affects the micellization process is through its effect on the structure of water.

Dynamical and structural properties of water/alcohol mixtures

Abstract Our recent studies related to the properties of alcohol/water mixtures, show the occurrence of some kind of molecular aggregation in the water rich region of composition beyond a threshold alcohol concentration x 2 ∗ . The observed behaviour suggests that for x 2 2 ∗ the alcohol molecules are essentially dispersed and surrounded by “water cages” where the short range order and microdynamic of water molecules are changed with respect to those of pure water. Alcohol molecules are in mutual contact at higher concentration, only, when almost all water is involved in hydration shells of alcohol molecules. In addition, these studies show that the stabilisation of micellar structure and nucleic acids conformation in water/alcohol mixtures is closely linked to the properties and anomalous behaviour of the solvent systems. Accordingly, these results support the hypothesis that the dominant mechanism by which an alcohol affects these processes is through its effect on the structure of water. The main results of these investigations are reviewed and discussed in this paper.

Dielectric Relaxation Spectroscopy of Lysozyme Aqueous Solutions: Analysis of the δ-Dispersion and the Contribution of the Hydration Water

The dielectric properties of lysozyme aqueous solutions have been investigated over a wide frequency range, from 1 MHz to 50 GHz, where different polarization mechanisms, at a molecular level, manifest. The dielectric relaxation spectra show a multimodal structure, reflecting the complexity of the protein-water interactions, made even more intricate with the increase of the protein concentration. The deconvolution of the spectra into their different components is not unambiguous and is generally a delicate process which requires caution. We have analyzed the whole relaxation region, on the basis of the sum of simple Debye-type relaxation functions, considering three main contributions. Particular attention has been payed to the δ-dispersion, intermediate between the β-dispersion (rotational dynamics of the protein) and the γ-dispersion (orientational polarization of the water molecules). This intermediate contribution to the dielectric spectrum is attributed to the orientational polarization of water molecules in the immediate vicinity of the protein surface (hydration water). Our measurements clearly demonstrate that, at least at high protein concentrations, the δ-dispersion has a bimodal structure associated with two kinds of hydration water, i.e., tightly bound and loosely bound hydration water. In the concentration range investigated, the existence of a three-modal δ-dispersion, as recently suggested, is not supported, on the basis of statistical tests, by the analysis of the dielectric relaxations we have performed and a bimodal dispersion is accurate enough to describe the experimental data. The amount of the hydration water has been evaluated both from the dielectric parameters associated with the δ-dispersion and from the decrement of the loss peak of the γ-dispersion. The relative weight of tightly bound and loosely bound hydration water is briefly discussed.

X‐ray absorption near edge structure (XANES) of Cu(II)–ATP and related compounds in solution: Quantitative determination of the distortion of the Cu site

We have studied the Cu binding site in Cu(II)–ATP, Cu(II)–tripolyphosphate, and Cu(II)–D‐ribose complexes in aqueous solution at several pH values ranging from 3 to 12 by means of high‐resolution x‐ray spectroscopy. We have measured the spectra in the x‐ray absorption near edge structure (XANES) region to probe the tetragonal distortion of the Cu site in these compounds, as indicated by optical d–d spectroscopy. The spectrophotometric studies proposed that Cu(II) bind to the oxygen atoms of the phosphate groups of ATP with a distorted octahedral symmetry at low pH, while at high pH, the metal atom would be bound to the hydroxyl groups of the ribose moiety with an almost planar symmetry. We have compared the measured XANES spectra with multiple scattering calculations, by using as a structural model a simple tetragonal CuO6 cluster with fixed equatorial distance (1.95 A) and variable axial distance (1.95–2.7 A). The multiple scattering calculations fit well the experimental data of Cu–ATP in solution, and ...

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.

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.

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.

INFLUENCE OF PH ON LYSOZYME CONFORMATION REVEALED BY DIELECTRIC SPECTROSCOPY

In this paper we report permittivity measurements in the frequency range 105–108 Hz, at the fixed temperature of 20°C, on lysozyme dissolved in water and in a water–ethanol mixture (ethanol concentration 0.1 molar fraction) varying the pH of the solutions from acid (≅pH 2) to basic (≅pH 10). The experimental data were fitted with the Cole–Cole equation and from the dispersion parameters the effective hydrodynamic radius and the electric dipole moment of the protein in different conditions of pH were calculated. The results confirm a conformational effect induced on lysozyme by ethanol, reported in our previous work. For pH in the range 4–6, where the enzymatic activity of lysozyme reaches its maximum, our results indicate a stable conformation of the protein. Out of this pH interval aggregation and expansion processes are present.