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Updated: May 18, 2026
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Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET
Published on: October 9, 2021
Nucleotide activation of the Ca-ATPase
Joseph M Autry1, John E Rubin, Bengt Svensson
1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
The Journal of Biological Chemistry
|September 15, 2012
Summary
We studied sarcoplasmic reticulum Ca-ATPase (SERCA) dynamics using fluorescence and modeling. We found headpiece closure couples to substrate binding, not active site dynamics, crucial for Ca(2+) binding and enzyme function.
Area of Science:
- Biochemistry
- Structural Biology
- Enzyme Kinetics
Background:
- Sarcoplasmic reticulum Ca-ATPase (SERCA) is crucial for muscle contraction by pumping calcium ions.
- Understanding SERCA's structural dynamics is key to elucidating its transport mechanism.
Purpose of the Study:
- To investigate the structural dynamics of SERCA, specifically the sarcoplasmic reticulum Ca-ATPase 1a (SERCA1a) isoform.
- To correlate headpiece closure with substrate binding and active site dynamics.
Main Methods:
- Fluorescence spectroscopy (lifetime, anisotropy, quenching) to probe protein dynamics.
- Molecular modeling to calculate solvent-accessible surface area.
- Limited proteolysis to assess conformational changes.
Main Results:
- FITC labeling of Lys-515 did not induce headpiece closure, indicating a nucleotide-free state.
- FMP formation induced headpiece closure, mimicking ATP binding.
- FMP-SERCA showed increased probe dynamics but decreased accessibility compared to FITC-SERCA.
- Headpiece closure is coupled to substrate binding, not active site dynamics.
Conclusions:
- SERCA headpiece closure is regulated by substrate binding.
- Dynamics within the nucleotide-binding site are vital for Ca(2+) binding and phosphoenzyme formation.
- These findings offer insights into SERCA's allosteric regulation and catalytic mechanism.
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