What Can Really Be Learned from Dielectric Spectroscopy of Protein Solutions? A Case Study of Ribonuclease A

Abstract

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.

Molecular View of Water Dynamics near Model Peptides

“…We find that the high-concentration data is consistent with peptide-driven motions of the first hydration layer, while the low-concentration data introduces a new reorientational motion that is solely due to hydration water dynamics, involving dynamic coupling between inner and outer hydration layers and consistent with the shorter time scale δ-dispersion. [42][43][44][45]47 Together, these results suggest that the faster component of the δ-relaxation could be made to disappear for proteins under severe hydration conditions. 47 Our QENS measurements and interpretation may also be consistent with those observed in the TDFSS profile (if relaxation due to the protein environment can be unambiguously removed).…”

“…[58][59][60][61][62][63][64][65] There is some disagreement as to whether the large range of time scales measured by these techniques, from tens of picoseconds to hundreds of nanoseconds, is actually directly attributable to the hydration layer nearest the protein or peptide surface or to outer hydration layers or even coupling of the hydration dynamics to different components of the solute motion. 18,47,66,67 Although there is agreement that the protein surface hydration layer dynamics are very heterogeneous, there is little information as to which components of the protein surface chemistry contribute to this heterogeneity. At present, a molecular interpretation is needed that would specify the chemical features of the protein surface, the distinct hydration layers ranging from protein surface waters to outer hydration layers to bulk liquid as well as the dynamics of the biological solute, to explain the large dynamic time scale range that is observed.…”

“…18 Attempts to explain the discrepancy between these techniques have given rise to a number of experimental studies and theoretical analysis. 18,46,47,63,66,73 Recently, time-resolved fluorescence spectroscopy, which measures a collective environmental response after electronic excitation of a fluorophore located near the protein surface, has been used to study the hydration water dynamics. Several solvation dynamics studies have been carried out using either an endogenous tryptophan residue in the protein as a probe or an extrinsic probe covalently attached to the protein.…”

Study of the charge dynamics in mineral oil under a non-homogeneous field

“…Frood and Gallagher discovered that the experimental results of the frequency response of liquid are in agreement with Coelho's theory only when an additional contribution to the permittivity arising from DC conductivity has been taken into account [37]. It has been reported that if the polarization induced from DC conductivity is added to the total polarization, a better fit to the experimental results can be achieved [37][38][39][40][41]. In our previous work, a model has been proposed for oil dielectric response in frequency response with two types of charge carriers being involved [42][43][44].…”

Self-consistent treatment of the local dielectric permittivity and electrostatic potential in solution for polarizable macromolecular force fields

“…Introduction). Traditional dielectric spectroscopy of protein solutions 56,57 shows that protein/water interfaces have unique dielectric behaviors, different from that of bulk water. Simulations have also shown that the average dielectric permittivity of interfaces tends to be lower than that of the bulk solution.…”