Hyper-mobile water is induced around actin filaments

Abstract

When introduced into water, some molecules and ions (solutes) enforce the hydrogen-bonded network of neighboring water molecules that are thus restrained from thermal motions and are less mobile than those in the bulk phase (structure-making or positive hydration effect), and other solutes cause the opposite effect (structure-breaking or negative hydration effect). Using a method of microwave dielectric spectroscopy recently developed to measure the rotational mobility (dielectric relaxation frequency) of water hydrating proteins and the volume of hydration shells, the hydration of actin filament (F-actin) has been studied. The results indicate that F-actin exhibits both the structure-making and structure-breaking effects. Thus, apart from the water molecules with lowered rotational mobility that make up a typical hydration shell, there are other water molecules around the F-actin which have a much higher mobility than that of bulk water. No such dual hydration has been observed for myoglobin studied as the representative example of globular proteins which all showed qualitatively similar dielectric spectra. The volume fraction of the mobilized (hyper-mobile) water is roughly equal to that of the restrained water, which is two-thirds of the molecular volume of G-actin in size. The dielectric spectra of aqueous solutions of urea and potassium-halide salts have also been studied. The results suggest that urea and I(-) induce the hyper-mobile states of water, which is consistent with their well-known structure-breaking effect. The molecular surface of actin is rich in negative charges, which along with its filamentous structure provides a structural basis for the induction of a hyper-mobile state of water. A possible implication of the findings of the present study is discussed in relation to the chemomechanical energy transduction through interaction with myosin in the presence of ATP.

FIGURE 1

Hydration numbers of proteins estimated using dielectric spectroscopy. The ordinate (N_total) is the number of hydrating water molecules per protein molecule calculated from the dielectric exclusion volume, and the abscissa (N_cal) is the solvent-accessible surface areas (ASA)-based estimate. Data were from Yokoyama et al. (2001) and Suzuki et al. (1997b), for which the line was drawn by a linear regression analysis excluding the unfilled circle denoting a value for F-actin on the basis of its monomer unit estimated in this study (see Table 1).

FIGURE 2

Dielectric spectra of actin and myoglobin solutions. Unfilled and solid symbols refer to the real and imaginary parts of dielectric spectra, respectively; blue symbols refer to buffers. (a) Spectra of actin (magenta triangles, 21.0 mg/ml) in the low-salt buffer (2 mM HEPES, 0.2 mM ATP, and 0.1 mM CaCl₂, at pH7.2). (b) Spectra of actin (red circles, 13.8 mg/ml) in the high-salt buffer (2 mM HEPES, 0.2 mM ATP, 0.1 mM CaCl₂, 50 mM KCl, and 2 mM MgCl₂, at pH 7.2). (c) Spectra of myoglobin (green triangles/stars, 14.0 mg/ml) in the low-salt buffer. (d) Spectra of myoglobin (black triangles/stars, 13.8 mg/ml) in the high-salt buffer.

FIGURE 3

Difference dielectric spectra of F-actin (red circles) and myoglobin (black squares) solutions. Data were obtained from eight independent measurements for both proteins, averaged as described in Materials and Methods, and normalized to a protein concentration of 10 mg/ml. The size of the spectral symbols are roughly equal to the error widths. Unfilled and solid symbols refer to the real and imaginary parts of dielectric spectra as in Fig. 2. (a) Difference spectra (Δɛ′ (f), Δɛ″(f)) to show that actin and myoglobin are distinct. The theoretical curves for myoglobin were calculated according to Yokoyama et al. (2001) and those for F-actin by analysis using the model shown in Fig. 5. (b) Comparison of fit of theoretical curves: the single-shelled model, green solid lines; the double-shelled model, blue solid lines. (c) Protein concentration-dependence of Δɛ′ (black) and Δɛ″ (red) of F-actin at the frequencies indicated.

FIGURE 4

Dielectric spectra of hydrated F-actin and myoglobin. Unfilled and solid symbols refer to the real and imaginary parts of dielectric spectra as in Fig. 2. (a) Comparison of actin (red circles) and myoglobin (black squares) in the high-salt buffer. (b) Comparison of actin in the low-salt buffer (magenta triangles) and the high-salt buffer (red circles). (c) Comparison of myoglobin in the low-salt buffer (green triangles) and the high-salt buffer (black squares).

FIGURE 5

Double-shelled ellipsoidal solute model. This model (Asami et al., 1980) was used to analyze the dielectric spectrum of F-actin solution as outlined in Materials and Methods. The expressions 2r_x and 2r_y refer to the lengths of major and minor axes of the ellipsoidal shells, respectively. Theoretical spectral curves obtained were superimposed on the observed values for Δɛ′(f) and Δɛ″(f) (blue-colored solid lines in Fig. 3 a). Note that when applied to the spectrum of the myoglobin solution with the protein axial ratio of 1.001, a curve essentially the same as estimated by the previous method was obtained (Yokoyama et al., 2001).

FIGURE 6

Dielectric spectra of hydrated solutes in urea and potassium halides solutions. (a) Experimental values for urea ( squares; circles) measured in 2% (w/v) solution are superimposed with theoretical curves (red and blue) simulated with the two components combined together: one with f_c1 = 6.5 GHz, δ₁ = 110, and N₁ = 2.4; and the other with f_c2 = 30GHz, δ₂ = 88, and N₂ = 2.4 using Eqs. 1–5 and N_1,2 = φ_1,2M_w/18c. The magenta-colored curve is the spectrum (imaginary part) of the component with f_c1 = 6.5 GHz. (b) Spectra of potassium halide salts, KF (black), KCl (red), and KI (blue) measured in 0.2 M solutions. The curves were calculated using Eq. 5 with a constant φ_t = 0.117, which was the largest among those of KF, KCl, and KI. The peak in spectra are at 18 GHz for KF, whereas the simulation indicates peaks at f_c2 = 22 GHz and 25 GHz for KCl and KI, respectively.

FIGURE 7

A diagrammatic representation of the idea underlying the hypothesis of unidirectional sliding of myosin along F-actin. (a) The surface of F-actin is covered by a layer of hyper-mobile water (for simplicity's sake, the ordinary restrained water is not shown). (b) Upon interaction with a specific binding protein, actin changes its conformation, which is in turn propagated directionally to adjacent monomers to generate solvent space of axially skewed viscosity. (c) During an ATP hydrolyzing cycle, myosin undergoes conformational changes in coupling with alternating strong and weak affinities for actin. The conformational changes are transmitted to actin and propagated as in b. Thus, the asymmetric space for diffusion is dynamically generated, where myosin moves toward the direction of lower viscosity.