Spectroscopic evidence for interaction between transmembrane helices 3 and 5 in rhodopsin
Spectroscopic evidence for interaction between transmembrane helices 3 and 5 in rhodopsin
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DOI：
10.1021/bi9801560
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发表时间：
1998-05-19
期刊：
影响因子：
2.9
通讯作者：
Siebert, F
中科院分区：
文献类型：
--
作者：
Beck, M;Sakmar, TP;Siebert, F
最近的视紫红质（Rho）分子模型提出了跨膜（TM）螺旋3和5之间的特异性相互作用，这似乎分别由TM螺旋3和5上的氨基酸残基Glu（122）和His（211）介导。为了测试这种提出的相互作用，制备了Rho的四个单位点组氨酸置换突变体（H100 N、H152 N、H211 N和H211 F）、两个单位点谷氨酸置换突变体（E122 Q和E122 A）和三个双位点置换突变体（E122 Q/H211 F、E122 Q/H211 N和E122 A/H211 F）。表达的突变体色素重组成膜进行了研究，特别是解决的过渡到metarhodopsin I（MI）的FTIR差光谱。结果表明，脂质环境的影响频带典型的MI状态。具有取代的Glu（122）的突变体的光谱允许在Rho的暗态和MI中质子化Glu（122）的C=O伸缩的归属。His（211）的突变，而不是其他组氨酸残基的突变，影响这些归属于Glu（122）的振动模式。此外，His（211）的取代影响蛋白质模式，这些模式被认为是由第三个羟基携带基团产生的，该基团也与Glu（122）相互作用。当突变体E122 A中Glu（122）被Ala取代时，这些模式也受到影响，但当突变体E122 Q中Glu（122）被Gln取代时，这些模式不受影响。这些结果为Rho中TM螺旋3和5之间的相互作用提供了直接的实验证据，该相互作用由Glu（122）和His（211）介导。
Recent molecular models of rhodopsin (Rho) propose a specific interaction between transmembrane (TM) helices 3 and 5, which appears to be mediated by amino acid residues Glu(122) and His(211) on TM helices 3 and 5, respectively. To test this proposed interaction, four single-site histidine replacement mutants (H100N, H152N, H211N, and H211F), two single-site glutamic acid replacement mutants (E122Q and E122A), and three double-site replacement mutants (E122Q/H211F, E122Q/H211N, and E122A/H211F) of Rho were prepared. The expressed mutant pigments reconstituted into membranes were studied by FTIR difference spectroscopy addressing especially the transition to metarhodopsin I (MI). It is shown that the lipid environment influences bands typical of the MI state. Spectra of mutants with substituted Glu(122) allowed assignments of the C=O stretch of protonated Glu(122) in the dark state and in MI of Rho. Mutation of His(211), but not of other histidine residues, affects these vibrational modes assigned to Glu(122)., addition, replacements of His(211) affect protein modes that are proposed to arise from a third, hydroxyl-bearing group, which also interacts with Glu(122). These modes are influenced as well when Glu(122) is replaced by Ala in mutant E122A but not when it is replaced by Gin in mutant E122Q. These results provide direct experimental evidence for an interaction between TM helices 3 and 5 in Rho, which is mediated by Glu(122) and His(211).