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Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle
Published on: March 21, 2025
713
Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering.
Harsha Ravishankar1, Martin Nors Pedersen2, Mattias Eklund1,2,3,4,5,6,7
1Department of Chemistry, Umeå University. Linnaeus Väg 10, 901 87 Umeå, Sweden.
Science Advances
|March 29, 2020
Summary
Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters
Area of Science:
- Biochemistry
- Structural Biology
- Membrane Proteins
Background:
- Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters are crucial for calcium signaling.
- Previous studies using X-ray crystallography characterized SERCA transport but missed key intermediate states.
- Understanding SERCA's dynamic transitions is vital for comprehending calcium regulation.
Purpose of the Study:
- To track the real-time dynamics of SERCA transport in a native membrane environment.
- To identify and characterize missing intermediate states in the SERCA reaction cycle.
- To elucidate the timing of domain rearrangements during calcium transport.
Main Methods:
- Combined time-resolved X-ray solution scattering (TR-XSS) experiments.
- Utilized molecular dynamics (MD) simulations for real-time tracking.
- Investigated SERCA dynamics in a native membrane setting.
Main Results:
- The equilibrium [Ca2+]E1 state exhibited domain arrangements distinct from crystal structures.
- A 1.5-ms intermediate state showed cytoplasmic domain closure upon ATP release.
- A subsequent 13-ms transient state revealed a novel actuator domain arrangement exposing the ADP-binding site.
Conclusions:
- TR-XSS and MD simulations successfully captured SERCA reaction cycle dynamics in real-time.
- Elucidated the relative timing of previously unresolved domain rearrangements during SERCA transport.
- Provided critical insights into the mechanism of calcium ion reuptake by SERCA transporters.
