Takao Kodama

Hyper-Mobile Water Is Induced around Actin Filaments

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.

Fractal dimension analysis and mathematical morphology of structural changes in actin filaments imaged by electron microscopy

In this work, we examined structural changes of actin filaments interacting with myosin visualized by quick freeze deep-etch replica electron microscopy (EM) by using a new method of image processing/analysis based on mathematical morphology. In order to quantify the degree of structural changes, two characteristic patterns were extracted from the EM images. One is the winding pattern of the filament shape (WP) reflecting flexibility of the filament, and the other is the surface pattern of the filament (SP) reflecting intra-molecular domain-mobility of actin monomers constituting the filament. EM images were processed by morphological filtering followed by box-counting to calculate the fractal dimensions for WP (D(WP)) and SP (D(SP)). The result indicates that D(WP) was larger than D(SP) irrespective of the state of the filament (myosin-free or bound) and that both parameters for myosin-bound filaments were significantly larger than those for myosin-free filaments. Overall, this work provides the first quantitative insight into how conformational disorder of actin monomers is correlated with the myosin-induced increase in flexibility of actin filaments along their length as suggested by earlier studies with different techniques. Our method is yet to be improved in details, but promising as a powerful tool for studying the structural change of protein molecules and their assemblies, which can potentially be applied to a wide range of biological and biomedical images.

Hydration properties of adenosine phosphate series as studied by microwave dielectric spectroscopy.

Hydration properties of adenine nucleotides and orthophosphate (Pi) in aqueous solutions adjusted to pH=8 with NaOH were studied by high-resolution microwave dielectric relaxation (DR) spectroscopy at 20 °C. The dielectric spectra were analyzed using a mixture theory combined with a least-squares Debye decomposition method. Solutions of Pi and adenine nucleotides showed qualitatively similar dielectric properties described by two Debye components. One component was characterized by a relaxation frequency (f(c)=18.8-19.7 GHz) significantly higher than that of bulk water (17 GHz) and the other by a much lower f(c) (6.4-7.6 GHz), which are referred to here as hyper-mobile water and constrained water, respectively. By contrast, a hydration shell of only the latter type was found for adenosine (f(c)~6.7 GHz). The present results indicate that phosphoryl groups are mostly responsible for affecting the structure of the water surrounding the adenine nucleotides by forming one constrained water layer and an additional three or four layers of hyper-mobile water.

Hepatocyte nuclear factor-4 alpha/gamma and hepatocyte nuclear factor-1 alpha as causal factors of interindividual difference in the expression of human dihydrodiol dehydrogenase 4 mRNA in human livers.

Human dihydrodiol dehydrogenase (DD) catalyses the oxidation of trans-dihydrodiols of polycyclic aromatic hydrocarbons and the reduction of several ketone-containing drugs. About 40-fold interindividual difference in DD activities has been noted. Recently, we found that transcriptional factors, hepatocyte nuclear factor (HNF)-1 alpha, HNF-4 alpha and HNF-4 gamma were essential for the expression of DD4 mRNA, which is a major form of DDs. Thus, to clarify a possible mechanism(s) underlying the interindividual difference in DD activities, we investigated the sequences of genes and the expression levels of mRNA for DD4 and HNFs in human livers. We found no clear relationship between the genotypes of DD4 and HNF genes and the expression levels of DD4 mRNA in the subjects. The expression level of DD4 mRNA significantly correlated with that of HNF-1 alpha, HNF-4 alpha or HNF-4 gamma. These results suggest that the expression level of DD4 mRNA is cooperatively regulated by the amounts of HNF-1 alpha, HNF-4 alpha and HNF-4 gamma.

Metabolic and energetic aspects of the growth response of Streptococcus rattus to environmental acidification in anaerobic continuous culture

Streptococcus rattus, a serotype b strain of mutans streptococci, was grown in an anaerobic glucose-limited chemostat. The molar growth yield of glucose [Yglucose, g dry wt (mol glucose)-1] together with the maximum growth yields (Ymax) and maintenance coefficients for glucose utilization and calculated ATP generation were estimated as a function of pH. When the pH was lowered from 7.0 to 5.0, Yglucose decreased, with a concomitant gradual change in the composition of the end product from a mixture of formate, acetate and ethanol to one mostly of lactate. Whereas the Ymax for glucose decreased without any change in the Ymax for ATP on acidification, both of the maintenance coefficients markedly increased. Kinetic and immunochemical examinations indicated the presence of an F1F0-type proton-translocating ATPase in the membrane fraction prepared from bacterial cells grown under acidic conditions; no detectable level of the enzyme was found in cells grown at neutral pH. However, when incubated with glucose under non-growing conditions, these acid-adapted and unadapted cells showed an insignificant difference in the ability to maintain the intracellular pH alkaline relative to the acidic environments. These results suggest that the organism responds and adapts to environmental acidification by sacrificing some energy cost in terms of both the efficiency of glucose utilization to generate ATP and the extra maintenance required to continue biomass production as efficiently as under neutral pH.

Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo 1H NMR and fluorescence spectroscopy

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo (1)H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ∼5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.

Kinetics of dextran-independent α-(1→3)-glucan synthesis by Streptococcus sobrinus glucosyltransferase I.

Glucosyltransferase (GTF)‐I from cariogenic Streptococcus sobrinus elongates the α‐(1→3)‐linked glucose polymer branches on the primer dextran bound to the C‐terminal glucan‐binding domain. We investigated the GTF‐I‐catalyzed glucan synthesis reaction in the absence of the primer dextran. The time course of saccharide production during dextran‐independent glucan synthesis from sucrose was analyzed. Fructose and glucose were first produced by the sucrose hydrolysis. Leucrose was subsequently produced, followed by insoluble glucan [α‐(1→3)‐linked glucose polymers] after a lag phase. High levels of intermediate nigerooligosaccharide series accumulation were characteristically not observed during the lag phase. The results from the enzymatic activity of the acceptor reaction for the nigerooligosaccharide with a degree of polymerization of 2–6 and methyl α‐d‐glucopyranoside as a glucose analog indicate that the activity increased with an increase in the degree of polymerization. The production of insoluble glucan was numerically simulated using the fourth‐order Runge–Kutta method with the kinetic parameters estimated from the enzyme assay. The simulated time course provided a profile similar to that of experimental data. These results define the relationship between the kinetic properties of GTF‐I and the time course of saccharide production. These results are discussed with respect to a mechanism that underlies efficient glucan synthesis.

A novel approach for purification of recombinant proteins using the dextran-binding domain.

Using the dextran‐binding domain (DBD) of a type of glucosyltransferase (GTF) from Streptococcus sobrinus, we have developed a novel method for purifying recombinant proteins. DBD‐tagged green and red fluorescent proteins as well as the parent GTF and DBD moiety were adsorbed well to commercially available cross‐linked dextran (such as Sephadex beads and Sephacryl beads), and eluted efficiently with water‐soluble dextran. The purity of the eluted proteins after this one‐step affinity purification was ∼90% or better. The results suggest that DBD can be used as a powerful carrier for purification of various recombinant proteins.

Single-Molecule Imaging of Interaction between Dextran and Glucosyltransferase from Streptococcus sobrinus

Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of Streptococcus sobrinus. Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constraints estimated for TMR-labeled GTF at pH 6.8 and 25 degrees C in the absence and presence of sucrose were 9.2 and 13.3 s(-1), respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (> or = 25 s(-1)) under comparable conditions.

Chemomechanical coupling in actomyosin system: An approach by in vitro movement assay and kinetic analysis of ATP hydrolysis by shortening myofibrils

On the basis of our recent studies of the sliding distance of actin filaments during one ATP cycle on the surface of myosin-coated glass surface and ATP hydrolysis by rapidly shortening myofibrils, the molecular mechanism of chemomechanical coupling is considered. We conclude that the myosin head can repeat many active cyclic interactions with actins to drive the actin filaments over a long distance during one ATP cycle, and that the distance is variable depending on the load.