摘要：The sarcoendoplasmic reticulum Ca
2+-ATPase (SERCA) transports Ca
2+ ions across the membrane coupled with ATP hydrolysis. Crystal structures of ligand-stabilized molecules indicate that the movement of actuator (A) domain plays a crucial role in Ca
2+ translocation. However, the actual structural movements during the transitions between intermediates remain uncertain, in particular, the structure of
E2PCa
2 has not been solved. Here, the angle of the A-domain was measured by defocused orientation imaging using isotropic total internal reflection fluorescence microscopy. A single SERCA1a molecule, labeled with fluorophore ReAsH on the A-domain in fixed orientation, was embedded in a nanodisc, and stabilized on Ni–NTA glass. Activation with ATP and Ca
2+ caused angle changes of the fluorophore and therefore the A-domain, motions lost by inhibitor, thapsigargin. Our high-speed set-up captured the motion during
EP isomerization, and suggests that the A-domain rapidly rotates back and forth from an
E1PCa
2 position to a position close to the
E2P state. This is the first report of the detection in the movement of the A-domain as an angle change. Our method provides a powerful tool to investigate the conformational change of a membrane protein in real-time.
