Mieko Taniguchi

Mechano-chemical behaviour of F-actin

Even at very low ionic strengths, actin assumes a polymerized form of moderate size under sonic vibration. This was deduced from the following facts: (a) rapid formation of long F-actin filaments takes place after sonication for a short period; (b) the actin shows moderately high viscosity at the time of stopping the vibration; and (c) actin placed in a sonic field is not denatured by EDTA. The short actin polymer formed by the vibration splits added ATP enzymically. After stopping the vibration, i.e. when the transformation of the short actin polymers into long F-actin filaments takes place, a large amount of ATP is split. The initial velocity of this ATP splitting is proportional to the actin concentration. This finding was explained by assuming that each short actin polymer contains an appreciable proportion of partially interrupted structures of the F-form and their reformation is coupled with the ATP splitting. From the re-examination of the facts obtained from the sonic experiments, the following conclusions were drawn. (1) The interruption-reformation process is essentially reversible; (2) interruption is accelerated by mechanical forces; (3) reformation is accelerated by the dephosphorylation of added ATP. Taking into account these results, various dynamic properties of an F-actin filament which theoretically follow from the helical polymer model are presented.

Conformational changes of F-actin in the thin filaments of muscle induced in Vivo and in Vitro by calcium ions

Abstract Ultraviolet linear dichroism measurements were undertaken to investigate conformational changes of F-actin in the thin filaments of muscle induced by calcium ions. The absorption of two polarized light beams was measured in a glycerinated single fiber of crab leg muscle, which has a very long sarcomere, using an optical microscope. The dichroism spectrum of I-band was very similar to that of an oriented gel of pure F-actin from the crab muscle; that is, it had two negative peaks (larger absorption of light polarized perpendicular to the fiber axis). One appeared at about 260 nm, which is due to ADP bound to actin, and the other at about 295 nm, which is probably due to a specific tryptophan residue in actin. These negative peaks of dichroism of the I-band became smaller in the presence of Ca 2+ than in its absence, both by about 30%. The dichroism measurement was also undertaken on the reconstituted F-actin-tropomyosin-troponin complex from rabbit muscle oriented by flow. The negative dichroism of the complex at about 260 nm and 295 nm became smaller in the presence of Ca 2+ than in its absence, both by about 20%. The magnitude of dichroism of F-actin in the complex in the presence of Ca 2+ was the same as that of F-actin alone. The decrease in dichroism is evidence for a change in the state of actin in the muscle fiber induced by Ca 2+ . It is caused either by a change of intramonomer conformation or intermonomer arrangement in F-actin or by increased fluctuation of monomer orientation. H-band (the non-overlapping region of A-band) gave positive dichroism between 260 nm and 300 nm with a peak at about 280 nm, which is due to tyrosine and tryptophan residues in the thick filament. This dichroism did not depend on the Ca 2+ concentration (in the absence of ATP). In addition, a remarkable difference was found in the dichroism around 280 nm between F-actins from rabbit and from crab, suggesting a difference in the structure of actin monomers.

The trimer-disc transformation of tobacco mosaic virus protein

Abstract The trimer of tobacco mosaic virus (TMV) protein can be reversibly transformed into a disc form, an intermediate state before the protein polymerizes into rods like native TMV. This trimer-disc transformation is separately regulated by the salt condition and temperature. By systematic kinetic and equilibrium analyses it is shown that the trimer-disc transformation is a kind of reversible condensation phenomenon, to which can be applied a theory developed on the polymerization-depolymerization of protein molecules by Oosawa and Higashi 1. The enthalpy change for the transformation is found to be about 77 kcal/mole.

Flow dichroic spectra of tobacco mosaic virus and their protein assemblies

Ultraviolet flow dichroisms of tobacco mosaic virus (TMV), TMV-RNA and TMV-protein were measured using three strains of TMV. 1. Large positive dichroisms were observed with three strains of TMV, namely ordinary, bean form of bean, and tomato strains (TMV-OM, TMV-B and TMV-T, respectively) at about 255, 276, 284 and 290 nm. The positive dichroisms were confirmed with reconstituted protein assemblies of TMV-OM and TMV-B at about 276, 284 and 290 nm where tyrosine and tryptophan residues of these proteins contribute. These results show that the electronic transition moments of their base groups and aromatic groups are nearly parallel to the polymer axis. It is suggested that there is a strong interaction between the base groups of RNA and aromatic groups of amino acid residues in TMV. 2. A small negative dichroism was observed near 296-300 nm with intact TMV-OM and TMV-T and with the reconstituted protein polymer of TMV-OM. But negative dichroism was not observed either with the intact virus or the protein assembly of TMV-B. 3. Isolated RNA from TMV-OM, TMV-B and TMV-T showed no dichroism. The configuration of RNA in TMV appears to be imposed on it by its packing with the protein.

Thermally induced conformational changes of tobacco mosaic virus and their protein assemblies

The measurement of the fluorescence intensity of tryptophan residues showed that a reversible transition in the local structure took place between 20 degrees C and 30c in intact virus particles and reconstituted protein assemblies of the ordinary strain and the tomato strain of tobacco mosaic virus. During this transition the overall polymer structure was maintained. In the case of the bean strain of tobacco mosaic virus, however, the fluorescence intensity did not show any transition in the same temperature range. Such a difference between different strains gave some information on the location of tryptophan residues possibly involved in the local structure change. The fluorescence polarization of intact virus particles showed no change in the whole temperature range, but the polarization of the reconstituted protein assembly of the ordinary strain showed a transition at the same temperature as the fluorescence intensity. This suggested a difference in the freedom of the local structure between intact virus particles and reconstituted protein assemblies. Oligomers of these virus proteins were stable up to 45c and above this temperature, began to make an irreversible transition where the secondary structure of the monomer was partially broken but the oligomer structure was retained.

Characterization of monomers and small assemblies of tobacco mosaic virus proteins using the electric birefringence method

Abstract 1. 1.|The electric birefringence of monomers and oligomers of proteins from the ordinary strain of tobacco mosaic virus (TMV-O) and from the bean strain of TMV (TMV-B) was measured using the high rectangular pulse method. 2. 2.|The specific Kerr constants for monomers of both proteins in 33% acetic acid were about 2·10 −8 e.s.u. The specific Kerr constant for the oligomer of the TMV-O protein in a 5 mM phosphate buffer (pH 7.0) was of the same order as for these monomers, while that for the TMV-B protein in the same solvent condition was two or three times larger (5.0·10 −8 e.s.u.). 3. 3.|The relaxation time of birefringence was about 0.025–0.030 μsec for monomers and about 0.140–0.150 μsec for oligomers. 4. 4.|The oligomer of the TMV-B protein was distinguished from that of the TMV-B protein by the contribution of the permanent dipole moment to the electric birefringence. Some qualitative difference between the proteins in the process of constructing oligomers from monomers was suggested.

Ultraviolet flow dichroism of brain microtubule.

Ultraviolet flow dichroism of brain microtubule was measured. Large positive dichroisms were observed at 255 nm, 285 nm and 292 nm. The dichroism at about 255 nm is due to bound GDP or GTP. The dichroism at 285 nm is due to tyrosine and tryptophan residues, and that at 292 nm is due to the tryptophan residues of the protein. These results show that the electronic transition moments of nucleotides and aromatic groups are both nearly parallel to the polymer axis.

Conformational change and behavior of tyrosine residues of bacterial flagella and flagellin at high pH

Abstract In order to investigate the coupling between intra- and intermolecular structure which may have some relation to the motility of flagella, the stability and structural changes of flagella and flagellin from Salmonella typhimurium were measured using optical rotatory dispersion, spectrophotometric titration, flow birefringence and electron microscopy. 1. 1. Alkali treatment brought about a conformational change in flagellin molecules which is regarded as a typical helix-coil transition. 2. 2. In flagellin a few tyrosine residues were exposed outside of the molecule and the remainder were buried inside. In flagella no tyrosines were exposed; namely, normal tyrosines in flagellin were buried inside of the polymer structure. 3. 3. The polymerizability could be recovered even after the flagellin was exposed to high pH where most of the tyrosine residues were titrated. 4. 4. No appreciable depolymerization of the flagella was observed, even when considerable amounts of buried tyrosine residues were dissociated.

Soft X-ray microscope with zone plate at UVSOR

Abstract A soft X-ray microscope with zone plates was set up at UVSOR [synchrotron radiation facility (750MeV, 200mA) at Institute for Molecular Science, Okazaki, Japan]. A 50nm line & space pattern could be resolved at λ=3.2nm. With an environmental chamber (wet cell) using SiN windows, wet biological specimens, such as letus protoplasts, rabbit myofibrils, tubulin of COS cell and Deinococcus radiodurans strains, could be observed at λ=2.5nm. In the present microscope, the numerical aperture of the condenser was much smaller than that of the objective. To adjust both the numerical apertures, an ellipsoidal condenser mirror system was tested, and preliminary result (an image of Cu mesh, 12.7μm pitch) was obtained.

Observation of wet biological specimen by soft X-ray microscope with zone plates at UVSOR

With an environmental chamber (wet cell) using polypropylene foils as windows, wet specimens were observed at a wavelength of 4.6 nm with a zone plate imaging X-ray microscope installed at the beamline 8 A of UVSOR (synchrotron radiation facility at Institute for Molecular Science, Okazaki, Japan). Images of spicule of trepang, human blood cells and cultured protoplast of plant cell stained by methyl mercury were observed with good contrast.