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Sites of labeling with N-(3-pyrene)maleimide on Ca(2+)-transporting ATPase of the sarcoplasmic reticulum
1Department of Physiological Chemistry, Tokyo Metropolitan Institute of Medical Science.
Abstract:
The characteristics of the interaction between N-(3-pyrene)maleimide (PMI) and the sarcoplasmic reticulum (SR) membranes were investigated, and the sites of labeling with PMI on Ca(2+)-transporting ATPase were identified. PMI was dissolved in the membrane lipids before reacting with the ATPase protein. The measurement of resonance energy transfer from PMI to 1-(dimethylaminophenyl)-6-phenyl-1,3,5-hexatriene revealed that the pyrene moiety of PMI stayed in the lipid layer after it had been covalently attached to the ATPase molecule. PMI-labeled SR membranes at an average labeling density of 1 mol PMI/mol ATPase were digested with trypsin, and the labeled peptides were purified through a series of reversed-phase HPLC procedures on C18 and C4 columns. The amino acid analysis of the purified peptides revealed multiple cysteine residues mainly distributed over the C-terminal half of the cytoplasmic domain of the ATPase molecule as the targets of PMI. This implied that PMI molecules mediated cross-linking between the cytoplasmic domain of the ATPase molecule and the membranes. The distortion of the structure of the former due to this cross-linking may explain the uncoupling of ATP-splitting from Ca(2+)-transport caused by PMI.
Insights
N-(3-pyrene)maleimide (PMI) binds to sarcoplasmic reticulum (SR) membranes, labeling cysteine residues on the Ca(2+)-transporting ATPase. This cross-linking distorts the ATPase structure, uncoupling ATP hydrolysis from calcium transport.
Area of Science:
- Biochemistry
- Membrane Biology
- Protein Chemistry
Background:
- Sarcoplasmic reticulum (SR) Ca(2+)-transporting ATPase is crucial for muscle contraction.
- Understanding protein-membrane interactions is key to elucidating enzyme function.
- N-(3-pyrene)maleimide (PMI) is a fluorescent probe used to study membrane protein interactions.
Purpose of the Study:
- Investigate the interaction between PMI and SR membranes.
- Identify specific labeling sites of PMI on the Ca(2+)-transporting ATPase.
- Elucidate the mechanism by which PMI affects ATPase function.
Main Methods:
- Resonance energy transfer measurements to track PMI localization.
- Proteolytic digestion (trypsin) of labeled SR membranes.
- Purification of labeled peptides using reversed-phase HPLC (C18 and C4 columns).
- Amino acid analysis to identify labeled cysteine residues.
Main Results:
- PMI's pyrene moiety remains in the lipid layer after covalent attachment to the ATPase.
- Multiple cysteine residues in the C-terminal half of the ATPase's cytoplasmic domain are labeled by PMI.
- PMI mediates cross-linking between the ATPase cytoplasmic domain and the membrane.
Conclusions:
- PMI labeling reveals specific interaction sites on the Ca(2+)-transporting ATPase.
- PMI-induced cross-linking likely causes structural distortions in the ATPase.
- These structural changes explain the observed uncoupling of ATP hydrolysis from Ca(2+) transport by PMI.