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

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 struct...

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Autores principales:	Takanobu A. Katoh, Takashi Daiho, Kazuo Yamasaki, Stefania Danko, Shoko Fujimura, Hiroshi Suzuki
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/42b4be887568440d87720bad745fb185
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spelling	oai:doaj.org-article:42b4be887568440d87720bad745fb1852021-12-02T14:34:02ZAngle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging10.1038/s41598-021-92986-32045-2322https://doaj.org/article/42b4be887568440d87720bad745fb1852021-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-92986-3https://doaj.org/toc/2045-2322Abstract 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.Takanobu A. KatohTakashi DaihoKazuo YamasakiStefania DankoShoko FujimuraHiroshi SuzukiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Takanobu A. Katoh Takashi Daiho Kazuo Yamasaki Stefania Danko Shoko Fujimura Hiroshi Suzuki Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging
description	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.
format	article
author	Takanobu A. Katoh Takashi Daiho Kazuo Yamasaki Stefania Danko Shoko Fujimura Hiroshi Suzuki
author_facet	Takanobu A. Katoh Takashi Daiho Kazuo Yamasaki Stefania Danko Shoko Fujimura Hiroshi Suzuki
author_sort	Takanobu A. Katoh
title	Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging
title_short	Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging
title_full	Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging
title_fullStr	Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging
title_full_unstemmed	Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging
title_sort	angle change of the a-domain in a single serca1a molecule detected by defocused orientation imaging
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/42b4be887568440d87720bad745fb185
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Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging

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