Strong Dependence of Hydration State of F-Actin on the Bound Mg²⁺/Ca²⁺ Ions

Understanding of the hydration state is an important issue in the chemomechanical energetics of versatile biological functions of polymerized actin (F-actin). In this study, hydration-state differences of F-actin by the bound divalent cations are revealed through precision microwave dielectric relaxation (DR) spectroscopy. G- and F-actin in Ca- and Mg-containing buffer solutions exhibit dual hydration components comprising restrained water with DR frequency f₂ (<f_w: DR frequency of bulk solvent, 17 GHz at 20 °C) and hypermobile water (HMW) with DR frequency f₁ (>f_w). The hydration state of F-actin is strongly dependent on the ionic composition. In every buffer tested, the HMW signal D_hyme (≡ (f₁ - f_w)₁/(f_ww)) of F-actin is stronger than that of G-actin, where _w is DR-amplitude of bulk solvent and ₁ is that of HMW in a fixed-volume ellipsoid containing an F-actin and surrounding water in solution. D_hyme value of F-actin in Ca2.0-buffer (containing 2 mM Ca²⁺) is markedly higher than in Mg2.0-buffer (containing 2 mM Mg²⁺). Moreover, in the presence of 2 mM Mg²⁺, the hydration state of F-actin is changed by adding a small fraction of Ca²⁺ (∼0.1 mM) and becomes closer to that of the Ca-bound form in Ca2.0-buffer. This is consistent with the results of the partial specific volume and the Cotton effect around 290 nm in the CD spectra, indicating a change in the tertiary structure and less apparent change in the secondary structure of actin. The number of restrained water molecules per actin (N₂) is estimated to be 1600-2100 for Ca2.0- and F-buffer and ∼2500 for Mg2.0-buffer at 10-15 °C. These numbers are comparable to those estimated from the available F-actin atomic structures as in the first water layer. The number of HMW molecules is roughly explained by the volume between the equipotential surface of -kT/2e and the first water layer of the actin surface by solving the Poisson-Boltzmann equation using UCSF Chimera.