Conformation of a peptide encompassing the proton
translocation channel of vacuolar H+-ATPase
Werner L. Vos 1, Louic S. Vermeer 1 and Marcus A. Hemminga 2*
1 Laboratory of Biophysics, Wageningen University
2 Biophysics Laboratory, Wageningen University
* To whom correspondence should be addressed. E-mail: marcus.hemminga{at}wur.nl.
Submitted on May 23, 2006
Revised on July 2, 2006
Accepted on 14 September 2006
 |
Abstract |
|---|
The structural properties of a crucial transmembrane helix for proton translocation in V-ATPase are studied using double site-directed spin labeling (SDSL) combined with electron spin resonance (ESR) (or electron paramagnetic resonance, EPR) and circular dichroism (CD) spectroscopy in SDS micelles. For this purpose, we use a synthetic peptide derived from TM7 of subunit a from the yeast Saccharomyces cerevisiae V-ATPase that contains two natural cysteine residues suitable for spin labeling. The interspin distance is calculated using a second moment analysis of the methanethiosulfonate spin label ESR
spectra at 150 K. Molecular dynamics (MD) simulation is used to study the effect of the sidechain dynamics and backbone dynamics on the interspin distance. Based on the combined results from ESR, CD, and MD simulation we conclude that the peptide forms a dynamic 
-helix. We discuss this finding in the light of current models for proton translocation. A novel role for a buried charged residue (H729) is proposed.
Key Words:
CD, ESR/EPR, SDSL, V-ATPase subunit a, molecular dynamics simulations, proton pump
