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.
Highlights
The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ ions across the membrane coupled with ATP hydrolysis
Single ReAsH-attached SERCA1a molecules were embedded in single nanodiscs of POPC (1-palmitoyl-2-oleoyl-glycero phosphatidylcholine)[24], and stabilized on the surface of Ni–NTA coated glass via two his-tags located in the nanodisc (Figs. 1a, 2c)
Fluorescent images showed that the number of bright spots decreases as the concentration of labeled SERCA1a is reduced and the bright spots completely disappear from the Ni–NTA glass with imidazole treatment (Fig. 2d,e and Supplementary Fig. S2), indicating that we are able to detect a signal from the ReAsH probe attached to the nanodiscembedded SERCA1a
Summary
The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ ions across the membrane coupled with ATP hydrolysis. 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. The A-domain appears to show significant changes in angle during the cycle[1,6,14] This conformational change in the A-domain is coupled with an alteration of Ca2+ affinity inside the membranous region and the opening and closing of a release-gate through the three linkers. Isomerization, Ca2+ ions are released to the luminal s ide[1,17] These findings are derived from static crystal structures and the actual domain movements between conformations of intermediates remain conjectural. Noteworthy, our high-speed set-up was capable of observing the back and forth motion of the A-domain during EP isomerization
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