1P169 Analysis of angular steps of bacterial flagellar motors using an elliptic probe(11.Molecular motor,Poster,The 51st Annual Meeting of the Biophysical Society of Japan)

Abstract

Kinesins are microtubule (MT) -based motor proteins that power intracellular transport. By using the 3-D tracking microscopy we develped, it was shown that MT rotates in a left-handed manner with the pitch of ∼0.3 μm when it slides on the lawns of single-headed kinesins. We here introduced mutations at the N-terminal region which was proposed to interact with the necklinker as a cover strand (CS) form to build a β-sheet. By tracking quantum dot attached to MT, the corkscrewing motion of MT driven by mutants were directly and quantitatively visualized in 3-D. The pitch depends on the type of mutations, indicating that one of the structural bases that causes torque is CS. 1P165 G-、F-アクチンの水和測定と偏比容測定Hydration and partial specific volume measurements of G-and The role of hydration water is a key issue in actin polymerization. We studied hydration properties and partial specific volume of actin by dielectric relaxation spectroscopy (DRS) and density measurements (DM). By DRS, constrained water (fc = 5.1 GHz at 10 °C) and hyper mobile water (HMW) (fc = 20 GHz) were detected around G-and F-actin. The HMW content was higher for F-actin than for G-actin. DMs of actin solutions showed that partial specific volumes of G-and F-actin were 0.715 ± 0.006 and 0.726 ± 0.003 ml/g at 10 °C, respectively. The thermal expansibilities were 6.8 × 10 Hydration water is vital to molecular processes involving proteins and has altered properties such as modulated local viscosity. Since rotational motion of a fluorophore reflects its surrounding viscosity, we have investigated fluorescence anisotropy of a fluorophore tethered to a protein.A frequency-domain fluorometer was in-house constructed to measure time resolved fluorescence anisotropy. Rhodamine 6G (R6G) was tethered to C374 of actin through oligo(ethylene glycol). Two components were found in the anisotropy decay of R6G and the rotational correlation times were 0.5 ns and 25 ns at 20°C, which would correspond to rotation of R6G and tumbling of actin, respectively. The former fast component is likely to be a measure of the hydration water viscosity around actin. Alkylureas modulate protein properties and enzymatic activities. Cosolvent-and drug-induced changes in actomyosin have been exploited to better understand its mechanism of force generation. In this study, we have investigated effects of alkylureas on actomyosin. Mg-ATPase of myosin subfragment-1 in the presence of urea, 1,3-dimethylurea, 1,3-diethylurea (DEU), or thiourea below 1 M showed that DEU enhanced the turnover rate by up to 7-fold whereas the others had a lesser effect on it. Interestingly, the maximal rate V max of actin-activated heavy meromyosin ATPase was almost unchanged with 0.3 M DEU, whereas the sliding speed of myosin-driven actin filaments was reduced to 1/16 of control. Thus, DEU effectively uncouples the sliding motility from actomyosin ATPase. The rotational angular steps of flagellar motor have been studied under very low ion motive force conditions in which rotation speed is unusually slow and unstable...