Detergent binding explains anomalous SDS-PAGE migration of membrane proteins
Detergent binding explains anomalous SDS-PAGE migration of membrane proteins
复制标题
DOI：
10.1073/pnas.0813167106
复制
发表时间：
2009-02-10
影响因子：
11.1
通讯作者：
Deber, Charles M.
中科院分区：
文献类型：
--
作者：
Rath, Arianna;Glibowicka, Mira;Deber, Charles M.
十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上的迁移与分子量无关，称为“凝胶移位”，似乎是膜蛋白的常见现象，但尚未得到最终解释。在目前的工作中，我们研究了异常凝胶流动性的螺旋膜蛋白使用的野生型和突变体螺旋-环-螺旋（“发夹”）序列的人囊性纤维化跨膜传导调节因子（CFTR）的跨膜段3和4，包括疾病表型残基取代的图书馆。我们发现，这些发夹在SDS-PAGE上以相对于其实际配方重量的-10%至+30%的速率迁移，并且以3.4-10 g SDS/g蛋白质的比率加载洗涤剂。我们还证明了突变体凝胶位移与发夹SDS加载能力（R-2 = 0.8）的变化密切相关，并与发夹螺旋度（R-2 = 0.9），表明凝胶位移行为起源于改变洗涤剂结合。在某些情况下，SDS的这种差异溶剂化作用可能是由于蛋白质-蛋白质接触取代了蛋白质-去污剂接触，这意味着去污剂结合和折叠密切相关。因此，我们文库中包含的CF表型V232 D突变体可能通过改变蛋白质-脂质相互作用破坏CFTR功能。观察到的发夹迁移，SDS聚集数和构象之间的相互依赖性，另外表明，去污剂结合可以提供一个快速和经济的筛选，用于识别具有强大的三级和/或四级结构的膜蛋白。
Migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that does not correlate with formula molecular weights, termed "gel shifting,'' appears to be common for membrane proteins but has yet to be conclusively explained. In the present work, we investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix ("hairpin'') sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. We find that these hairpins migrate at rates of -10% to +30% vs. their actual formula weights on SDS-PAGE and load detergent at ratios ranging from 3.4-10 g SDS/g protein. We additionally demonstrate that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity (R-2 = 0.8), and with hairpin helicity (R-2 = 0.9), indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked. The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. The observed interdependence between hairpin migration, SDS aggregation number, and conformation additionally suggests that detergent binding may provide a rapid and economical screen for identifying membrane proteins with robust tertiary and/or quaternary structures.