Structure and function in rhodopsin: Topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops
Structure and function in rhodopsin: Topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops
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DOI：
10.1073/pnas.94.26.14267
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发表时间：
1997-12-23
影响因子：
11.1
通讯作者：
Khorana, HG
中科院分区：
文献类型：
--
作者：
Cai, KW;Langen, R;Khorana, HG
半胱氨酸诱变和定位自旋标记的视紫红质的C-末端区域已被用来探测局部结构和接近该区域的细胞质环。序列T335-T340中的每个天然氨基酸被Cys替换，一次一个。所有突变体的巯基与巯基试剂4，4 '-二硫代二吡啶快速反应，这表明溶剂可及性程度高。此外，为了探索邻近关系，构建了一系列双Cys突变体。所有组中的一个Cys位于位置338，另一个位于EF螺旋间环中序列S240-V250中的位置，AB螺旋间环中的位置65，或CD螺旋间环中的位置140。在黑暗状态下，在所用条件下，在C338和C65或C140之间未观察到显著的二硫化物形成，而在C338和C242或C245之间观察到相对快速的二硫化物形成。在双半胱氨酸突变体的自旋标记显示最强的磁相互作用的氮氧化物连接到C338和C245或C246之间。光激活的双突变体T242 C/S338 C导致较慢的二硫键形成，而在C338和C245或C246的氮氧化物之间的相互作用减少。这些结果表明，邻近的C-末端残基C338的F螺旋的细胞质螺旋延伸的外表面上的残基与光活化后的距离明显增加。
Cysteine mutagenesis and site-directed spin labeling in the C-terminal region of rhodopsin have been used to probe the local structure and proximity of that region to the cytoplasmic loops. Each of the native amino acids in the sequence T335-T340 was replaced with Cys, one at a time. The sulfhydryl groups of all mutants reacted rapidly with the sulfhydryl reagent 4,4'-dithiodipyridine, which indicated a high degree of solvent accessibility. Furthermore, to probe the proximity relationships, a series of double Cys mutants was constructed. One Cys in all sets was at position 338 and the other was at a position in the sequence S240-V250 in the EF interhelical loop, at position 65 in the AB interhelical loop, or at position 140 in the CD interhelical loop. In the dark state, no significant disulfide formation was observed between C338 and C65 or C140 under the conditions used, whereas a relatively rapid disulfide formation was observed between C338 and C242 or C245. Spin labels in the double Cys mutants showed the strongest magnetic interactions between the nitroxides attached to C338 and C245 or C246. Light activation of the double mutant T242C/S338C resulted in slower disulfide formation, whereas interactions between nitroxides at C338 and C245 or C246 decreased. These results suggest the proximity of the C-terminal residue C338 to residues located on the outer face of a cytoplasmic helical extension of the F helix with an apparent increase of distance upon photoactivation.