Structural features and light-dependent changes in the sequence 59-75 connecting helices I and II in rhodopsin: A site-directed spin-labeling study
Structural features and light-dependent changes in the sequence 59-75 connecting helices I and II in rhodopsin: A site-directed spin-labeling study
复制标题
DOI：
10.1021/bi990014l
复制
发表时间：
1999-06-22
期刊：
影响因子：
2.9
通讯作者：
Hubbell, WL
中科院分区：
文献类型：
--
作者：
Altenbach, C;Klein-Seetharaman, J;Hubbell, WL
在牛视紫红质细胞质表面跨膜螺旋I和II之间的序列56-75中制备了21个单半胱氨酸取代突变体。每个突变体与巯基特异性试剂反应以产生氮氧侧链。分析十二烷基麦芽糖苷溶液中标记蛋白质的电子顺磁共振，以提供极性和非极性顺磁试剂的氮氧侧链的相对迁移率和可及性。结果表明，胶束的疏水-水界面相交的螺旋I和II附近的残基64和71，分别。因此，序列64-71在水相中，而56-63和72-75分别位于跨膜螺旋I和II中。细胞质表面附近的跨膜螺旋I和II上的面向脂质的表面分别对应于螺旋表面上的大约180度和90度。视紫红质的光活化产生的结构变化，在该地区的调查，主要是围绕螺旋II。然而，这些变化比螺旋VI中的自旋标记所指出的变化小得多。
Twenty-one single-cysteine substitution mutants were prepared in the sequence 56-75 between transmembrane helices I and II at the cytoplasmic surface of bovine rhodopsin. Each mutant was reacted with a sulfhydryl-specific reagent to produce a nitroxide side chain. The electron paramagnetic resonance of the labeled proteins in dodecyl maltoside solution was analyzed to provide the relative mobility and accessibility of the nitroxide side chain to both polar and nonpolar paramagnetic reagents. The results indicate that the hydrophobic-water interface of the micelle intersects helices I and II near residues 64 and 71, respectively. Thus, the sequence 64-71 is in the aqueous phase, while 56-63 and 72-75 lie in the transmembrane helices I and II, respectively. The lipid-facing surfaces on transmembrane helices I and II near the cytoplasmic surface correspond to approximately 180 degrees and 90 degrees of are on the helical surfaces, respectively. Photoactivation of rhodopsin produced changes in structure in the region investigated, primarily around helix II. However, these changes are much smaller than those noted by spin labels in helix VI.