The bacterial flagellar motor is an ion-driven rotary machine in the cell envelope of bacteria. Using a gold nanoparticle as a probe, we observed the precession of flagella during rotation. Since the mechanism of flagella precession was unknown, we investigated it using a combination of full simulations, theory, and experiments. The results show that the mechanism can be well explained by fluid mechanics. The validity of our theory was confirmed by our full simulation, which was utilized to predict both the filament tilt angle and motor torque from experimental flagellar precession data. The knowledge obtained is important in understanding mechanical properties of the bacterial motor and hook.

Membrane proteins play a critically important role, especially in molecular and cellular transport processes, which makes them the most prevalent drug targets and also to be a good sensing element for biosensing applications. However, the skill-dependent and manual fashion to form bilayer lipid membranes (BLMs) has limited their use for application purposes. Here, we introduce a fabrication technique of SU-8 microchannel suitable for microfluidic-based BLM formation and show a preliminary step of our ISFET-based BLM sensor. Making notches on the corners of a rectangular through-wafer hole etched in a silicon carrier substrate reduced the gap width between an embedded chip and the carrier as close as possible, leading to the gap depth of 4 μm. SU-8 microchannel on the chip-carrier assembly was directly fabricated by UV exposure through a bonded quartz lid and developing SU-8 through the fluidic ports drilled in the lid, without needing a sealing process typically causes an alignment mismatch or void formation. Finally, the capability to form BLMs was tested and proven by the successfully encapsulated fluorescent dye in microwells fabricated on the embedded chip. This paper is the first report of the successful formation of BLMs on a chip-incarrier assembly by the microfluidic method.

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...

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There are emerging needs for bilayer lipid membrane (BLM) sensors utilizing the vastly and highly developed molecular recognition ability of membrane proteins, especially those combined with CMOS chips. However, this type of BLM sensor has barely been studied so far mainly because of two problems: (i) the difficulty of their fabrication processes resulting from chip-carrier discontinuity, (ii) a skill-dependent and manual fashion to form BLMs. Here, we introduce a gapless chip-in-carrier integration process utilizing parylene as a gap filler and a microfluidic packaging method with an injectable Ag/AgCl-epoxy reference electrode. These methods allow for subsequent photolithography processes and later microfluidic-based BLM formation where the surface continuity of a microchannel is crucial. As a proof-of-concept device, we fabricated chip-in-carrier assemblies with a dummy chip coupled with a microfluidic system for BLM formation compatible with simultaneous optical observation. With this device, we validated the capability to form BLMs by microfluidic technique and characterized the configuration of a formed BLM through the observation of fluorescent localization. This chip integration and microfluidic packaging scheme can provide a solution for the full implementation of CMOS ICs with BLM sensors and broaden the possibilities of practical applications in the engineering and medical fields.