Shin Yamaguchi

Dielectric Relaxations in Various Crystal Modifications of Cyclohexanol and 2,3-Dimethylbutane

Abstract The dielectric constant and loss of crystalline cyclohexanol (CHOL) and 2,3-dimethylbutane (DMB) were measured from 4°K to the melting point, 299°K for CHOL and 145°K for DMB. The supercooled plastic crystal of CHOL above the glass temperature Tg exhibits a prominent relaxation (α) due to molecular rotation. The temperature dependence of therelaxation time was found to obey the Fogel-Tamman equation. A secondary relaxation (β) was observed below Tg , 149°K for CHOL and 76°K for DMB. The mechanism proposed for β is internal rotation; rotationof OH bond for CHOL and trans-gauche transformation for DMB. The β relaxation of DMB is well correlated with acoustic relaxation in the liquid state in the relaxation time versus timperature plot. Relaxations in the intermediate state, crystal III, of these two substances and the most stable state, crystal II of DMB were studied.

Axonemal dynein light chain-1 locates at the microtubule-binding domain of the γ heavy chain.

Dynein light chain 1 (LC1) of the outer arm dynein (OAD) complex associates with the microtubule-binding domain (MTBD) of γ heavy chain inside the complex. LC1 is considered to regulate the OAD activity and ciliary/flagellar motion by modulating γ MTBDs affinity to the B-tubule of the doublet microtubule in the axoneme.

Torque Generation by Axonemal Outer-Arm Dynein

Outer-arm dynein is the main engine providing the motive force in cilia. Using three-dimensional tracking microscopy, we found that contrary to previous reports Tetrahymena ciliary three-headed outer-arm dynein (αβγ) as well as proteolytically generated two-headed (βγ) and one-headed (α) subparticles showed clockwise rotation of each sliding microtubule around its longitudinal axis in microtubule corkscrewing assays. By measuring the rotational pitch as a function of ATP concentration, we also found that the microtubule corkscrewing pitch is independent of ATP concentration, except at low ATP concentrations where the pitch generated by both three-headed αβγ and one-headed α exhibited significantly longer pitch. In contrast, the pitch driven by two-headed βγ did not display this sensitivity. In the assays on lawns containing mixtures of α and βγ at various ratios, the corkscrewing pitch increased dramatically in a nonlinear fashion as the ratio of α in the mixture increased. Even small proportions of α-subparticle could significantly increase the corkscrewing pitch of the mixture. Our data show that torque generation does not require the three-headed outer-arm dynein (αβγ) but is an intrinsic property of the subparticles of axonemal dyneins and also suggest that each subparticle may have distinct mechanical properties.

Circular orientation fluorescence emitter imaging (COFEI) of rotational motion of motor proteins

Single-molecule fluorescence polarization technique has been utilized to detect structural changes in biomolecules and intermolecular interactions. Here we developed a single-molecule fluorescence polarization measurement system, named circular orientation fluorescence emitter imaging (COFEI), in which a ring pattern of an acquired fluorescent image (COFEI image) represents an orientation of a polarization and a polarization factor. Rotation and pattern change of the COFEI image allow us to find changes in the polarization by eye and further values of the parameters of a polarization are determined by simple image analysis with high accuracy. We validated its potential applications of COFEI by three assays: 1) Detection of stepwise rotation of F1-ATPase via single quantum nanorod attached to the rotary shaft γ; 2) Visualization of binding of fluorescent ATP analog to the catalytic subunit in F1-ATPase; and 3) Association and dissociation of one head of dimeric kinesin-1 on the microtubule during its processive movement through single bifunctional fluorescent probes attached to the head. These results indicate that the COFEI provides us the advantages of the user-friendly measurement system and persuasive data presentations.