Conformation of a peptide encompassing the proton translocation channel of vacuolar H(+)-ATPase.

The structural properties of a crucial transmembrane helix for proton translocation in vacuolar ATPase are studied using double site-directed spin-labeling combined with electron spin resonance (ESR) (or electron paramagnetic resonance) and circular dichroism spectroscopy in sodium dodecyl sulfate micelles. For this purpose, we use a synthetic peptide derived from transmembrane helix 7 of subunit a from the yeast Saccharomyces cerevisiae vacuolar proton-translocating 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 simulation is used to study the effect of the side-chain dynamics and backbone dynamics on the interspin distance. Based on the combined results from ESR, circular dichroism, and molecular dynamics simulation we conclude that the peptide forms a dynamic alpha-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.