Actin: volume change on transformation of G-form to F-form.

The volume change occurring on polymerizing actin was measured by dilatometry. A large positive value of + 391 ml/mole was obtained for the volume change during the transformation of G- to F-actin. This large increase in volume could be interpreted as arising from the local change in the ordered water structure on the protein's surface at polymerizing sites.

The excimer fluorescence of N-(1-pyrenyl)iodoacetamide labeled to myosin and its subfragment 1.

Myosin and its subfragment 1 were labeled with the fluorescent probe N-(1-pyrenyl)iodoacetamide. Both of the labeled complexes exhibited the excimer band at 480 nm (pH 8.0, 25 degrees C). SH1 and SH2 are labeled with this probe as judged by Ca2+-ATPase of the labeled complex. Excimers arise both from the interaction of PIAAs in the two different heads within a single myosin molecule and also from the interaction of PIAAs in the same head. ATP affects these excimers depending on the concentration of Ca2+.

Excimer fluorescence to monitor transitions of the association dissociation equilibria induced by dilution of proteins composed of subunits.

Dilution is a simple method to induce shifts of the association-dissociation equilibria of protein subunits. Excimer fluorescence is useful to monitor changes of equilibria in dilute solution, which has been shown in a well-characterized protein, actin. Actin was labeled with a fluorescent probe; N-(1-pyrenyl)iodoacetamide. When actin was diluted in low ionic strength buffer, the relative excimer fluorescence decreased. This arose from the conformational drift of actin, which resulted in a change in the association-dissociation equilibria of actin subunits in the dilute solution. Since dilution effects are often observed in other proteins, excimer fluorescence would be a useful sensitive technique to detect shifts of association-dissociation equilibria in dilute solution.

Excimer fluorescence as a tool for monitoring protein domain dynamics applied to actin conformation changes based on circulary polarized fluorescence spectroscopy.

Fluorescence-detected circular dichroism (FDCD) was introduced into the study of protein conformation changes. Actin was used as a model protein which undergoes dynamic conformation changes as it polymerizes. Actin labeled with N-(1-pyrene)iodoacetamide (PIA) showed monomer fluorescence peak at 386 and 410 nm, and excimer fluorescence peak at around 480 nm. Excimer was formed by PIA-dimers labeled to different sites of amino acid residues. New information concerned with actin structural changes were monitored by fluorescence emission spectra excited with left- and right-circulary polarized light at 355 nm. FDCD intensities were shown as the difference in the fluorescence emission DeltaF, where DeltaF=(F (L)-F (R))/(F (L)+F (R)) denoting F (L) and F (R) as emissions obtained by excitation with left- and right-circulary polarized light. When solvent conditions of PIA-actin were changed by addition of NaCl, TFE, or ATP, DeltaF showed sensitive responses to these compounds. From the analysis of DeltaF (M) and DeltaF (E) which represent the peaks of DeltaF at the monomer- and excimer-emission band, the information concerned with the actin intrastructural changes were obtained. This method based on monitoring the excimer fluorescence with FDCD could be used for other proteins to extract finer structural changes that cannot be detected by the normal fluorescence spectroscopy.

Polymerization of actin induced by a molar excess of ATP in a low salt buffer.

The polymerization of actin induced by dilution has previously been reported, where a 1000-fold molar excess of ATP over actin resulted when actin was diluted to 4.0 microg/ml in low salt buffer A (0.1 mM ATP, 0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 5 mM 2-mercaptoethanol, 1 mM NaN3). Filaments formed by the addition of ATP to a 1000-fold molar excess over actin in buffer B (0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 1 mM NaN3) were then separated by gel-filtration. When ATP was removed from these filaments using Dowex-1, depolymerization occurred. Thus, the reversible polymerization induced by the dilution of actin or by addition of ATP can be ascribed to the binding of ATP at the low affinity site of actin.

Polymerization of actin induced by a molar excess of ATP in a low salt buffer.

The polymerization of actin induced by dilution has previously been reported, where a 1000-fold molar excess of ATP over actin resulted when actin was diluted to 4.0 micrograms/ml in low salt buffer A (0.1 mM ATP, 0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 5 mM 2-mercaptoethanol, 1 mM NaN3). Filaments formed by the addition of ATP to a 1000-fold molar excess over actin in buffer B (0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 1 mM NaN3) were then separated by gel-filtration. When ATP was removed from these filaments using Dowex-1, depolymerization occurred. Thus, the reversible polymerization induced by the dilution of actin or by addition of ATP can be ascribed to the binding of ATP at the low affinity site of actin.

Dilution beyond a transition concentration and the enhanced filaments' formation of actin in the low ionic strength buffer.

Actin in the low ionic strength buffer solution G (0.1 mM ATP, 0.1 mM CaCl2, 2 mM Tris-Cl (pH 8.0), 5 mM 2-mercaptoethanol and 1 mM NaN3) was diluted to the concentration below 10 micrograms/ml at 7 degrees C. Viscometry showed associations of actin monomers and increasing the extent of dilution, maximally about 75% of actin were precipitated. Actin filaments were observed by electron microscope in the diluted solution. Ca2+ plays a role in the filaments' formation. It seems that a phase transition like change in the actin states arose at this concentration range.

Anisotropy decay of labelled actin. Evidence of the flexibility of the peptide chain in F-actin molecules.

G actin, labelled presumably on cysteine-373 with the fluorescent chromophore N-iodoacetyl-N'-(5 sulfo-1-napthyl)-ethylenediamine and purified by Sephacryl S-200 gel chromatography, migrated in one band on polyacrylamide gel electrophoresis and had the same polymerizability as unlabelled purified G actin. Anisotropy decays of labelled actin solutions have been studied at different ionic strengths and protein concentrations. It was found that these anisotropy decays could be fitted by a sum of two exponential functions. Under low ionic strength or below the critical concentrations the longer correlation time (45 ns at 3.5 degrees C) was independent of protein concentration and ionic strength. Above the critical concentration, the longer correlation time increased with ionic strength and protein concentration. In order to take into account that, under these conditions, the solutions contained a mixture of F and G actin at the critical concentration, the anisotropy decays were analysed as a sum of three exponential functions in which the longest correlation time characterized F actin. Since F actin correlation time also depended on actin concentration, an analysis with a sum of four exponential functions was performed, in which two fixed correlation times (100 ns and 900 ns at 3.5 degrees C) were introduced in order to characterize the F actin motions. The lower of these correlation times was attributed to regions where two actin filaments interact side by side, while the shorter one was attributed to filament regions free from intermolecular interactions. The small value of the free F actin correlation time indicates that the protomer peptide chain is very flexible around its C terminus, probably involving the motion of a molecular lobe. This flexibility might be an important factor in the interaction of actin with myosin during the muscular contraction.

Cinemicrographic analysis of the movement of flagellated bacteria. II. The ratio of the propulsive velocity to the frequency of the wave propagation along flagellar tail.

We took cinemicrographs of the movement of the flagellar tail of the peritrichously flagellated bacterium, Salmonella, swimming in a medium containing methylcellulose under a dark-ground microscope. By analysing the film, the velocity of translation u, the frequency of the propagation of helical waves along the tail fF and the frequency of the induced rotation of bacterial body fB of individual organism were measured and the experimental values of the ratios u/fF, u/fB and fF/fB were obtained. On the other hand, the theoretical values of these ratios were calculated by inserting the geometrical parameters describing the shapes and the sizes of the body and the tail of individual organism into the equations previously derived for the hydrodynamic model of the propulsion of flagellated bacteria (Holwill and Burge, 1963; Chwang and Wu, 1971). For four bacterial specimens presently analysed, the experimental values of u/fF ranged from 0.5 to 0.9, whereas the theoretical values were about 0.3. As reported by the preceding paper, such a tendency for the experimental values to exceed the theoretical ones by two or three times was also seen in u/fB and, consequently, the experimental and the theoretical values of fF/fB showed good agreement. From the results of these quantitative analyses of the movements of flagellated bacteria, it was concluded that the validity of the hydrodynamic model was further supported experimentally.