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Conformational Ensembles from Experimental Data
and Computer Simulations
Poster Abstracts
128
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.