Conformational Ensembles from Experimental Data and Computer Simulations

Conformational Ensembles from Experimental Data and Computer Simulations

Poster Abstracts

91-POS Board 11 Single-Molecule Recordings of Gating Motions of KcsA Potassium Channels at Submillisecond Time Resolution Hirofumi Shimizu . University of Fukui, Yoshida-gun, Fukui, Japan. Until now, structural analyses of ion-channel proteins have shown static pictures at atomic resolutions. However, these pictures had no time stamps, and their structural stabilities were obscure. In this work, we adopted the diffracted X-ray tracking method for KcsA potassium channels to show time-stamped dynamic pictures of the conformational changes in the form of a movie. Here, the channel was fixed on a glass plate at the extracellular side in an upside-down orientation, while a gold nanocrystal was attached to the cytoplasmic side as a probe. The synchrotron white X-ray beam was directed perpendicular to the sample plate as the observation light, while a diffraction spot from the attached nanocrystal was tracked through the two- dimensional X-ray detector. In this geometry, the motions of the spots were translated into those of the channels; the circumferential and radial motions indicated the twisting and bending motions of the channels, respectively. Although we previously reported the global twisting motions upon gating of the KcsA channels at video rate, the time resolution was insufficient to reveal the entire picture of the gating motions. To resolve this problem, we recently introduced an X-ray focusing mirror and a high-speed X-ray detector system to the SPring8; these components enabled us to record the motions with wide spatial range at a submillisecond time resolution. By using this refined measurement system, the gating motions were recorded continuously in real time, providing information on the stabilities of the structures in transition states upon gating. The high spatial and temporal resolutions enabled the evaluation of single molecular fluctuations and conformational changes. In this session, we will present recent our data, which is expected to provide a contribution to the integrative structural biology of potassium channels.

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