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Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle
Published on: March 21, 2025
Substrate-induced conformational changes in sarcoplasmic reticulum Ca2+-ATPase probed by surface modification using
Ryouhei Narumi1, Taibo Yamamoto, Akio Inoue
1Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
FEBS Letters
|July 10, 2012
Summary
We identified 15 surface residues on the sarcoplasmic reticulum Ca(2+)-pump ATPase using mass spectrometry. Ligand binding, like ATP and Ca(2+), induced conformational changes in specific domains.
Area of Science:
- Biochemistry
- Structural Biology
- Membrane Proteins
Background:
- The sarcoplasmic reticulum Ca(2+)-pump ATPase (SERCA) is crucial for muscle contraction.
- Understanding its conformational dynamics upon ligand binding is key to its function.
Purpose of the Study:
- To identify surface-exposed residues of SERCA and investigate their reactivity changes upon binding of Ca(2+), ATP, and MgF(4).
- To correlate residue reactivity with structural and functional properties.
Main Methods:
- Mass spectrometry using diethylpyrocarbonate modification.
- Analysis of residue reactivity under varying conditions (ligand presence/absence).
- Correlation with atomic structure, solvent accessibility, pK(a), and hydrophobicity.
Main Results:
- 15 surface residues were identified; 9 showed consistently high reactivity.
- Lys-515 reactivity was inhibited by ATP; Lys-158 reactivity varied with Ca(2+) and MgF(4).
- Reactivity changes of 4 residues near domain interfaces suggested significant conformational shifts upon ligand binding.
Conclusions:
- Ligand binding induces distinct conformational changes in SERCA domains, particularly at inter-domain interfaces.
- Residue reactivity is a sensitive indicator of these structural dynamics.
- Atomic-level structural information explains observed reactivity patterns.
