Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging

The sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) transports Ca²⁺ 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 Ca²⁺ translocation. However, the actual structural movements during the transitions between intermediates remain uncertain, in particular, the structure of E2PCa₂ 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 Ca²⁺ 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 E1PCa₂ 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.