2P149 Protein-Protein Effective Interaction : Interaction between Attractive Patches Mediated by Multivalent Cations(11. Molecular motor,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

Abstract

Kinesin-1 is a motor protein that moves along microtubules in a hand-overhand manner. Recent crystal structure solved in our lab showed that kinesin head undergoes ~25 degree rotational motion of a subdomain upon ATP binding. In this study, we directly observed ATP-dependent rotational motion of the subdomain during processive motility using gold nanorod attached to one of the head. The centroid position and the angle of the nanorod were determined by fitting the focused dark-field image recorded at 100 μs temporal resolution to simulated PSF, and demonstrated that the angle of the nanorod showed two state transition while the labeled head binds to microtubule. We will also show the results of kinetics analysis and discuss the implication for the gating mechanism. 2P146X 線 1 分子計測による分子モーターの構造揺らぎ測定 X-ray Single Molecule Observations of Structural Fluctuations in Molecular MotorsKeigo Ikezaki 1 , Naruki Hara 1 , Hiroshi Sekiguchi 2 , Naoto Yagi 2 , Yuji Sasaki 1Molecular motors alter their molecular structures and exert mechanical force using the chemical energy of ATP hydrolysis. So for, many researchers revealed structural changes of molecular motors during ATP hydrolysis by using optical microscopy and atomic force microscopy. However, no one succeeds to clarify inter molecular fluctuations during ATP hydrolysis because of the resolution limit of their methods. Recently, we are trying to observe inter molecular fluctuations of myosin molecular motors using DXT measurement (described below). At the meeting, we will report the latest progress. Please look forward to it! Note: Diffracted X-ray Tracking DXT allows us to know the orientations of gold nano-crystals attached to protein surfaces.(SPring-8,BL40XU) Myosin IXb is a single-headed processive motor. Recent studies from two groups reported that the motility direction of myosin IXb was to either barbed end or point end direction. This discrepancy may arise from different sources of myosin or methods of actin preparation. Therefore, we hypothesized that the motility of myosin IXb is highly sensitive to subtle differences in actin structure. In this study, we first established a reproducible baculovirus-dependent system to express motile human myosin IXb. Movement of this myosin on Cy3-actin was to the barbed end, but interestingly, phalloidin was slightly inhibitory (0.66-fold slower). We will report the motile properties of several other types of actin filaments on this myosin. current across membrane down the electrochemical potential gradient is thought to drive the rotary motion of c-ring relative to a-subunit. As a molecular mechanism of the rotary motion, the half-channel model is widely accepted, where the two different H + channels at the interface of a-subunit and c-ring are assumed to play a key role. To investigate this model, we conducted all-atom molecular dynamics simulation, and analyzed water distribution in the membraneembedded region. We further studied the inter-subunit motion between cring and a-subunit. 2P149 タンパク質間の実行相互作用：多価カチオンを介した引力 パッチ間相...