Giuseppe Graziano

On the molecular origin of cold denaturation of globular proteins

A polypeptide chain can adopt very different conformations, a fundamental distinguishing feature of which is the water accessible surface area, WASA, that is a measure of the layer around the polypeptide chain where the center of water molecules cannot physically enter, generating a solvent-excluded volume effect. The large WASA decrease associated with the folding of a globular protein leads to a large decrease in the solvent-excluded volume, and so to a large increase in the configurational/translational freedom of water molecules. The latter is a quantity that depends upon temperature. Simple calculations over the -30 to 150 °C temperature range, where liquid water can exist at 1 atm, show that such a gain decreases significantly on lowering the temperature below 0 °C, paralleling the decrease in liquid water density. There will be a temperature where the destabilizing contribution of the polypeptide chain conformational entropy exactly matches the stabilizing contribution of the water configurational/translational entropy, leading to cold denaturation.

Hydration thermodynamics of aliphatic alcohols

The hydration thermodynamics of five linear aliphatic alcohols in the temperature range 5–100°C is carefully analysed using a suitably modified version of the theoretical approach developed by Lee. The hydration Gibbs energy change is determined by the balance of three contributions: the direct alcohol–water van der Waals interaction energy, the direct alcohol–water H-bond energy, and the excluded volume effect due to solute insertion. The analysis shows that the direct alcohol–water H-bond energy is fundamental in determining the negative values of the hydration Gibbs energy over the whole temperature range investigated, whereas the excluded volume effect determines the large and negative hydration entropies. The reorganization of H-bonds in the hydration shell of aliphatic alcohols proves to be a compensating process, not affecting the Gibbs energy change, as in the case of the hydration of nonpolar molecules. However, H-bond reorganization is the main molecular origin of the large and positive hydration heat capacity change, a signature of hydrophobic hydration, determining the temperature dependence of the hydration enthalpy and entropy changes. We show that H-bond reorganization can be reliably described by means of the modified Mullers model, indicating that the hydration shell is not akin to an iceberg: hydration shell H-bonds are energetically slightly stronger but more broken than those in bulk water. This finding allows the rationalization of the puzzling experimental data on the temperature dependence of the water proton NMR chemical shift in solutions of aliphatic alcohols.

On the temperature dependence of hydration thermodynamics for noble gases

The hydration thermodynamics of five noble gases in the temperature range 0–60°C have been carefully determined. We analyse these data using the theoretical approach developed by Lee that emphasizes that there are different physical causes for the large and positive hydration Gibbs energy change and the large and positive hydration heat capacity change. The analysis confirms that the hydrophobicity of noble gases is caused by the excluded volume effect due to solute insertion and exaggerated by the small size of water molecules at any temperature. The reorganization of H-bonds in the hydration shell of noble gases is a compensating process that does not contribute to the Gibbs energy change, but it is the cause of the large and positive hydration heat capacity change and determines the temperature dependence of the hydration enthalpy and entropy changes. The modified Mullers model, despite its simplicity, proves able to satisfactorily describe the reorganization of H-bonds, indicating that the hydration shell does not resemble an iceberg. The H-bonds in the hydration shell are energetically slightly stronger but more broken than those in the bulk water.

A van der Waals approach to the entropy convergence phenomenon

The van der Waals approach for the excluded volume effect is used to calculate the thermodynamics of cavity creation in a liquid. By using the experimental density of c-hexane, benzene and water and a temperature independent hard sphere diameter for solvent molecules, the van der Waals approach leads to the following results: (a) the values of the work of cavity creation show a linear decrease in the two organic solvents, but a parabolic temperature dependence with a maximum around 170°C in water; (b) the cavity entropy changes are positive and practically independent of temperature in the two organic solvents; (c) the cavity entropy changes in water are large negative at room temperature and then increase showing the convergence phenomenon at 160°C. Therefore, the experimental density of solvents at each temperature and the hard sphere diameter of solvent molecules considered to be temperature independent are sufficient to distinguish water from c-hexane and benzene and to reproduce entropy convergence.

On the effect of low concentrations of alcohols on the conformational stability of globular proteins

Low concentrations of alcohols have proven to be able to enlarge the stability curve of globular proteins, by decreasing the cold denaturation temperature and increasing the hot denaturation temperature [S. R. Martin, V. Esposito, P. De Los Rios, A. Pastore and P. A. Temussi, J. Am. Chem. Soc., 2008, 130, 9963-9970]. In order to try to explain these data, I have considered that: (1) an aqueous 2 M MeOH solution can be treated as a uniform liquid, constituted by water molecules, whose density, above the temperature of maximum density, has the same values of neat water, simply shifted by 2 °C toward lower temperatures, whereas, below the temperature of maximum density, it decreases to a slightly lesser extent than the density of neat water; (2) the ΔE(a)(2 M MeOH) quantity, a balance between intra-protein energetic attractions and those with the surrounding solvent molecules, both water and methanol, assumes a constant positive value. These physically-based assumptions, when inserted into the theoretical model developed to rationalize the occurrence of cold denaturation in neat water [G. Graziano, Phys. Chem. Chem. Phys., 2010, 12, 14245-14252], reproduce in a qualitatively correct manner the effect of low concentrations of alcohols.