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.
Vos
Werner L
WL
Laboratory of Biophysics, Wageningen University, Wageningen, The Netherlands.
Vermeer
Louic S
LS
Hemminga
Marcus A
MA
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D001692:Biological Transport; D001703:Biophysics / Q000379:methods*; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013696:Temperature; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry*
D010455:Peptides; D011522:Protons; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases