We report on a dielectric relaxation study of aqueous solutions of ribonuclease A at 298.15 K as a function of protein concentration between 0.5 and 6 wt % in the MHz/GHz frequency range. The spectra can be decomposed into five modes of Debye type diffusive behavior. In agreement with the standard interpretation, we assign the two dominant modes at low and high frequency (β-relaxation and γ-relaxation, respectively) to protein tumbling and bulk water relaxation. We observe three further modes (δ1−δ3) between β- and γ-relaxation, in contrast to a bimodal δ-dispersion frequently reported. We attribute the high frequency part (δ3) near 40 ps to hydration water reorientation, which, in the notion of other authors, corresponds to “loosely bound water”. We argue that the existence of “tightly bound” water, often deduced from the low frequency part in the nanosecond regime (δ1), is inconsistent with a highly mobile hydration layer observed by NMR techniques and molecular dynamics (MD) simulations. On the same grounds, we reject hydration water−bulk water exchange as a mechanism for δ-dispersion. In accordance with MD simulations, we assume that protein−water cross-correlations drive the nanosecond (δ1) process. We also discuss the role of intraprotein motions, which may contribute near 500 MHz (δ2). We discuss the meaning of the hydrodynamic radius and of the hydration numbers in light of the high mobility of hydration waters. We show that because of protein−protein interactions, the effective dipole moment of the protein decreases with increasing protein concentration.

A change in solvent can have dramatic effects on the physico-chemical properties of a protein and its stability. In this paper we demonstrate by a study of solutions of the enzyme ribonuclease A (RNase A) in normal water (H 2 O) and deuterated water (D 2 O), to what extend a solvent isotopic substitution affects the structural and dynamic properties of a protein and its stability. Differential scanning calorimetry (DSC) indicates a shift of the transition temperature from the native to the unfolded state from about 62 C in H 2 O to 66 C in D 2 O. Pressure perturbation calorimetry (PPC), a relatively new and efficient technique, is used to study the volumetric properties of RNase A in its native and unfolded state. In PPC, the coefficient of thermal expansion of the partial volume of the protein, a, is deduced from the heat consumed or produced after small isothermal pressure jumps (AE5 bar). a and its temperature coefficient, da/dT, strongly depend on the interaction of the protein with the solvent at the protein-solvent interface. Both quantities are markedly affected by H 2 O/D 2 O substitution. Dielectric spectroscopy in the MHz and GHz regime is used to characterize the H 2 O/D 2 O isotope effect upon the tumbling time and dipole moment of the protein. The analysis of the isotope effects gives evidence for a decrease of the dipole moment and hydrodynamic radius of the protein in D 2 O. An intriguing result is that the observed changes in thermodynamic properties reflect not only a stronger and more compact hydration in D 2 O, but also an increase in protein compactness. A similar result is obtained from dielectric relaxation experiments on another small globular protein, ubiquitin.

In the present study we combine dielectric relaxation spectroscopy with generalized Born simulations to explore the role of orientational order for protein aggregation in solutions of bovine pancreatic insulin at various pH conditions. Under aggregation-prone conditions at low pH, insulin monomers prefer antiparallel dipole alignments, which are consistent with the orientation of the monomeric subunits in the dimer structure. This alignment is also true for two dimers, suggesting that already at moderate protein concentrations the species assemble in equilibrium clusters, in which the molecules adopt preferred orientations also found for the protomers of the corresponding oligomers.