Structure and function in rhodopsin: Kinetic studies of retinal binding to purified opsin mutants in defined phospholipid-detergent mixtures serve as probes of the retinal binding pocket

被引:51
作者
Reeves, PJ
Hwa, J
Khorana, HG
机构
[1] MIT, Dept Biol, Cambridge, MA 02139 USA
[2] MIT, Dept Chem, Cambridge, MA 02139 USA
关键词
G-protein-coupled receptors; signal transduction; 11-cis-retinal; binding pocket; retinitis pigmentosa;
D O I
10.1073/pnas.96.5.1927
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
In the current standard procedure for preparation of mammalian rhodopsin mutants, transfected COS-1 cells expressing the mutant opsin genes are treated with 5 mu M 11-cis-retinal before detergent solubilization for purification. We found that binding of 11-cis-retinal to opsin mutants with single amino acid changes at Trp-265 (W265F,Y,A) and a retinitis pigmentosa mutant (A164V) was far from complete and required much higher concentrations of 11-cis-retinal. By isolation of the expressed opsins in a stable form, kinetic studies of retinal binding to the opsins in vitro have been carried out by using defined phospholipid-detergent mixtures. The results show wide variation in the rates of 11-cis-retinal binding. Thus, the in vitro reconstitution procedure serves as a probe of the retinal-binding pocket in the opsins. Further, a method is described for purification and characterization of the rhodopsin mutants after retinal binding to the opsins in vitro.
引用
收藏
页码:1927 / 1931
页数:5
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