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

Abstract

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.

Hydrogen Bond Network of Water around Protein Investigated with Terahertz and Infrared Spectroscopy

“…In other words, the relaxation modes of the albumin solute and hydration water, as well as the vibrational modes of albumin need to be taken into consideration. According to a recent study by Cametti et al (29), however, hydration water has two small relaxation modes at~0.1 and 5 GHz, thus the relaxation process of hydration water is negligibly small between 0.25 and 12 THz. Similarly, the relaxation mode of albumin solutes contributes little to the dielectric responses in the THz region, since the relaxation of protein molecules is located in the MHz region (29).…”

“…According to a recent study by Cametti et al (29), however, hydration water has two small relaxation modes at~0.1 and 5 GHz, thus the relaxation process of hydration water is negligibly small between 0.25 and 12 THz. Similarly, the relaxation mode of albumin solutes contributes little to the dielectric responses in the THz region, since the relaxation of protein molecules is located in the MHz region (29). In combination with our results in the previous section, both the relaxation and vibration modes of the albumin solute itself are considered negligibly small between 0.25 and 12 THz.…”

“…By doing so, the experimental uncertainty as well as the standard deviation in the fitting procedure is reflected in the best-fitted result. Additionally, even in the protein solution, bulk water molecules that are unperturbed by a solute molecule reorient at the same rate with those in pure water (29,31). Therefore, to obtain reliable fitting results, t slow was fixed to 7.93 ps for all the samples, which is equal to t slow of pure water at 300 K (75), and the remaining 10 parameters in Eq.…”

“…where C sol is the molar concentration of albumin solutes (29). The hydration number of albumin N hyd is summarized in Fig.…”

“…Molecular dynamics (MD) simulations (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) and other experimental approaches (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) have revealed the protein is surrounded by dynamically retarded hydration water, with the innermost shell having a density higher than that of bulk water. However, even today, experimentally characterizing the dynamics and the structure of the water HB network in this hydration shell is challenging, because the water-water HB lifetime is very short (typically 1 ps) (38).…”