STRUCTURE-FUNCTION STUDIES ON BACTERIORHODOPSIN .7. ASPARTIC-ACID SUBSTITUTIONS AFFECT PROTON TRANSLOCATION BY BACTERIORHODOPSIN
STRUCTURE-FUNCTION STUDIES ON BACTERIORHODOPSIN .7. ASPARTIC-ACID SUBSTITUTIONS AFFECT PROTON TRANSLOCATION BY BACTERIORHODOPSIN
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DOI：
10.1073/pnas.85.12.4148
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发表时间：
1988-06-01
影响因子：
11.1
通讯作者：
KHORANA, HG
中科院分区：
文献类型：
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作者：
MOGI, T;STERN, LJ;KHORANA, HG
我们已经替换了细菌视紫红质中的每个天冬氨酸残基，以确定它们在该蛋白质的质子转运中的可能作用。天冬氨酸残基被天冬酰胺取代，天冬氨酸-85、-96、-115和-212被谷氨酸取代，天冬氨酸212也被丙氨酸取代。将突变的细菌视紫红质基因在大肠杆菌中表达，并对蛋白进行纯化。当用洗涤剂-磷脂混合物和视网膜处理时，突变蛋白都能再生出类似细菌视紫红质的发色团。然而，生色团的再生速度和它们的最大波长变化很大。没有得到对视素移位的外部点电荷模型的支持。Asp-85.fwdarw。Asn突变体没有检测到质子泵送，Asp-96.fwdarw。Asn和Asp-212.fwdarw.Glu突变体显示-lt；10%，Asp-115.fwdarw。Glu突变体表现为apprxeq。是正常质子抽运的30%。讨论了这些发现对细菌视紫红质可能的质子转移机制的影响。
We have substituted each of the aspartic acid residues in bacteriorhodopsin to determine their possible role in proton translocation by this protein. The aspartic acid residues were replaced by asparagines; in addition, Asp-85, -96, -115, and -212 were changed to glutamic acid and Asp-212 was also replaced by alanine. The mutant bacteriorhodopsin genes were expressed in Escherichia coli and the proteins were purified. The mutant proteins all regenerated bacteriohodopsin-like chromophores when treated with a detergent-phospholipid mixture and retinal. However, the rates of regeneration of the chromophores and their .lambda.max varied widely. No support was obtained for the external point charge model for the opsin shift. The Asp-85 .fwdarw. Asn mutant showed no detectable proton pumping, the Asp-96 .fwdarw. Asn and Asp-212 .fwdarw. Glu mutants showed < 10% and the Asp-115 .fwdarw. Glu mutant showed .apprxeq. 30% of the normal proton pumping. The implications of these findings for possible mechanisms of proton translocation by bacteriorhodopsin are discussed.