Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging.
Sci Rep, 2021/07/01;11(1):13672.
Katoh TA[1, 2], Daiho T[3], Yamasaki K[3], Danko S[3], Fujimura S[4, 5], Suzuki H[6]
Affiliations
PMID: 34211016DOI: 10.1038/s41598-021-92986-3
Impact factor: 4.996
Abstract
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.
MeSH terms
Adenosine Triphosphate; Animals; COS Cells; Calcium; Chlorocebus aethiops; Models, Molecular; Protein Domains; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases
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