1P162 Analysis of the ATP-waiting form of F_1-ATPase by single-pair FRET measurement(11.Molecular motor,Poster,The 51st Annual Meeting of the Biophysical Society of Japan)
1P162 1分子FRET計測によるF_1-ATPaseのATP結合待ち構造の解析(11.分子モーター,ポスター,日本生物物理学会年会第51回(2013年度))

Mitsuhiro Sugawa, Masaru Kobayashi, Takashi Matsui, Tomoko Masaike
2013 Seibutsu Butsuri  
Mycoplasma mobile has hundreds of legs approximately 100 nm long and glides on the substrate at the speed of 2.5 μm/sec. It is hypothesized that a cycle of attachment to and detachment from surface-immobilized sialyllactose (SL) drives the force of the gliding. To reveal the mechanism of directed movement as the sum of the force in each leg, we quantified the interaction between legs and SL by using optical tweezers in the presence of different nucleotides or analogues: AMP-PNP ; ADP; ADP+Vi ;
more » ... ucleotide-free condition. We found that unbinding force depends on chemical states, and also, loading directions in terms of a bacterium gliding polarity. These nucleotide-dependent asymmetric variations could contribute to decipher the gliding mechanism at the molecular level. 1P159 滑走するバクテリアの戦車のような運動装置を三次元で追跡 する Three-dimensional tracking of tank-like motility apparatus of the gliding bacterium Showko Odaka, Daisuke Nakane, Takayuki Nishizaka (Department of Physics, Gakushin University) Flavobacterium johnsoniae exhibits rapid gliding motility over surfaces. The cell does not have flagella or pili, and instead, rely on a novel motility apparatus, the cell-surface adhesion, by a unique mechanism for efficient gliding. The latest contribution showed that the adhesions flow travels along a closed loop on a cell surface like a caterpillar in a tank (Nakane et al., PNAS, in press). Here we extend this approach and apply the threedimensional tracking method to the flow by nanometer-scale localization of 0.1 μm fluorescent beads to visualize the motion of adhesions on gliding cells. This method reveals the accurate flow pathway and its directionality: the left-handed helical closed loop with the helix of 3 μm in the rotational pitch and of 0.4 μm in radius. 1P160 戦車のような仕組みで動くバクテリア Flavobacterium johnsoniae exhibits rapid gliding motility over surfaces by a unique mechanism. These cells do not have flagella or pili; instead, they rely on a novel motility apparatus. SprB, a 669 kDa cell-surface adhesin, is required for efficient gliding. Here, we showed dynamic movements of SprB were observed by fluorescent microscopy. SprB moved at a constant speed of 2 μm/s on the cell surface along a left-handed helical closed loop, appears that the cell have a moving conveyer belt. Attachment of SprB to the substratum was associated with cell movement, suggesting a model for gliding, in which adhesins are propelled along a helical track, generating rotation and translation of the cell. 1P161 方位と倒れの構造変化を1分子レベルで検出する偏光スイッ チングを用いた新しい TIRFM Advanced TIRF microscopy to detect single-molecule conformational changes in both azimuth and axial axis using polarization switching To elucidate domain motions that correlate with protein functions at the single molecular level, we have developed objective-type TIRF microscopes with polarization modulation. We here extend this technique to detect not only conformational changes in azimuth but also tilting motions in axial angle, i.e., the component parallel to the optical axis, by switching the laser polarization. A pair of images under s-and p-polarized illuminations is continuously captured while spots from single fluorophores are rotated on the camera plate in synchronously. By fitting the intensity profile of each image, orientation of surface-immobilized catalytic subunit of F 1 -ATPase was detected with an angular resolution of ∼1° under the exposure of 800 millisecond. 1P162 分子 FRET 計測による F -ATPase の ATP 結合待ち構造の 解析 To uncover conformational changes in F 1 -ATPase, we performed singlepair FRET measurement to detect distance changes between subunits having a FRET pair (Cy3 and Cy5), using the mutant F1(βE190D/L398C). FRET signal showed at least two-state transition between high (~0.8) and low (~0.5) FRET efficiencies. The high FRET state represents the two β subunits taking a closed form. We also performed a simultaneous measurement of FRET and rotational steps. The high FRET state occurred at one of the three catalytic dwells. In other dwells, FRET efficiency remained lower. These results suggest that in the ATP-waiting dwell two of three β subunits should not take the closed form as in the catalytic dwell. 1P163 F 1 -ATPase の軸とシリンダーの結合寿命の測定 Measurement of lifetime of the bond between the shaft and the cylinder in single F 1 -ATPase
doi:10.2142/biophys.53.s132_5 fatcat:4vyktmgwabdkziqgs36reum5z4