Vos Werner L, Vermeer Louic S, Hemminga Marcus A
Laboratory of Biophysics, Wageningen University, Wageningen, The Netherlands.
Biophys J. 2007 Jan 1;92(1):138-46. doi: 10.1529/biophysj.106.089854. Epub 2006 Oct 13.
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.
利用双位点定向自旋标记结合电子自旋共振（ESR，或电子顺磁共振）以及在十二烷基硫酸钠胶束中的圆二色光谱，研究了液泡ATP酶中质子转运关键跨膜螺旋的结构特性。为此，我们使用了一种源自酿酒酵母液泡质子转运ATP酶a亚基跨膜螺旋7的合成肽，该肽含有两个适合自旋标记的天然半胱氨酸残基。通过对150 K下甲硫基磺酸盐自旋标记ESR光谱进行二次矩分析来计算自旋间距离。分子动力学模拟用于研究侧链动力学和主链动力学对自旋间距离的影响。基于ESR、圆二色性和分子动力学模拟的综合结果，我们得出该肽形成动态α螺旋的结论。我们根据当前的质子转运模型讨论了这一发现。提出了一个埋藏带电残基（H729）的新作用。
