Lindholm Ljubica, Ariöz Candan, Jawurek Michael, Liebau Jobst, Mäler Lena, Wieslander Åke, von Ballmoos Christoph, Barth Andreas
Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
Biochim Biophys Acta. 2015 Aug;1847(8):698-708. doi: 10.1016/j.bbabio.2015.04.011. Epub 2015 Apr 25.
The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-β-d-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli.
研究了特定脂质对细菌视紫红质质子泵视紫质（pR）的意义。为此，研究了pR是否优先结合某些脂质以及脂质环境的分子特性是否影响光循环。通过在可见光谱范围内的微秒级闪光光解跟踪pR的光循环。在磷脂酰胆碱脂质体（大豆脂质）中最快，在3-[(3-胆酰胺丙基)二甲基铵基]丙烷磺酸盐（CHAPS）：1,2-二油酰基-sn-甘油-3-磷酸胆碱（DOPC）双分子层和Triton X-100中居中，而当pR溶解在CHAPS中时最慢。在具有不同脂质组成的双分子层中，头部基团的性质、不饱和度水平和脂肪酸链长度对光循环影响较小。通过使用两种不同浓度的去污剂N-十二烷基-β-D-麦芽糖苷（DDM）从纯化的膜蛋白中分离脂质的优化方法，研究了pR对表达宿主大肠杆菌脂质的特异性亲和力。我们发现，在0.1% DDM时，每个pR分子共纯化出11种脂质，而在1% DDM时，基本上所有脂质都从pR中去除。共纯化的磷脂酰乙醇胺、磷脂酰甘油和心磷脂的相对含量与大肠杆菌细胞中通常存在的摩尔百分比无关。结果表明，重组pR周围脂质环中磷脂酰乙醇胺种类占优势，其极性低于表达宿主大肠杆菌细胞膜中的优势种类。
