视紫红质的结构与功能：在特定磷脂 - 去污剂混合物中，对纯化的视蛋白突变体与视黄醛结合进行动力学研究，以此作为视黄醛结合口袋的探针。
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.
作者信息
Reeves P J, Hwa J, Khorana H G
机构信息
Departments of Biology and Chemistry, Massachusetts Institute of Technology 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
出版信息
Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):1927-31. doi: 10.1073/pnas.96.5.1927.
Abstract
In the current standard procedure for preparation of mammalian rhodopsin mutants, transfected COS-1 cells expressing the mutant opsin genes are treated with 5 microM 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.
摘要
在目前制备哺乳动物视紫红质突变体的标准程序中，表达突变视蛋白基因的转染COS-1细胞在去污剂溶解以进行纯化之前，先用5 microM 11-顺式视黄醛处理。我们发现，11-顺式视黄醛与色氨酸-265（W265F、Y、A）处有单个氨基酸变化的视蛋白突变体以及一个视网膜色素变性突变体（A164V）的结合远未完成，并且需要更高浓度的11-顺式视黄醛。通过以稳定形式分离表达的视蛋白，利用确定的磷脂-去污剂混合物对视网膜与视蛋白的体外结合进行了动力学研究。结果显示11-顺式视黄醛结合速率存在很大差异。因此，体外重组程序可作为视蛋白中视网膜结合口袋的探针。此外，还描述了一种在视网膜与视蛋白体外结合后对视紫红质突变体进行纯化和表征的方法。
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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.视紫红质的结构与功能：在特定磷脂 - 去污剂混合物中，对纯化的视蛋白突变体与视黄醛结合进行动力学研究，以此作为视黄醛结合口袋的探针。
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本文引用的文献
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Prog Nucleic Acid Res Mol Biol. 1998;59:1-34. doi: 10.1016/s0079-6603(08)61027-2.
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