Department of Physics, Faculty of Science, Gakushuin University, Toshima-ku, Tokyo, 171-8588, Japan.
Laboratory for Organismal Patterning, RIKEN Center for Biosystems Dynamics Research, RIKEN, Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan.
Sci Rep. 2021 Jul 1;11(1):13672. doi: 10.1038/s41598-021-92986-3.
The sarcoendoplasmic reticulum Ca-ATPase (SERCA) transports Ca 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 translocation. However, the actual structural movements during the transitions between intermediates remain uncertain, in particular, the structure of E2PCa 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 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 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.
肌浆网 Ca2+-ATP 酶（SERCA）通过与 ATP 水解偶联将 Ca2+跨膜转运。配体稳定的分子的晶体结构表明，执行器（A）结构域的运动在 Ca 转运中起着关键作用。然而，在中间体之间的转变过程中实际的结构运动仍然不确定，特别是 E2PCa 的结构尚未解决。在这里，通过各向同性全内反射荧光显微镜的离焦取向成像测量 A 结构域的角度。将标记有 A 结构域上的荧光团 ReAsH 的单个 SERCA1a 分子以固定取向嵌入纳米盘，并稳定在 Ni-NTA 玻璃上。用 ATP 和 Ca 激活会引起荧光团和因此 A 结构域的角度变化，抑制剂 thapsigargin 会使运动失去。我们的高速装置捕捉到 EP 异构化过程中的运动，并表明 A 结构域从 E1PCa 位置快速前后旋转到接近 E2P 状态的位置。这是首次报道检测 A 结构域作为角度变化的运动。我们的方法为实时研究膜蛋白的构象变化提供了有力的工具。
