ROLE OF HELIX EMBEDDED PROLINE RESIDUES IN PHOTOCYCLE OF BACTERIORHODOPSIN

已有研究表明，X-脯氨酸肽键起了关键作用 膜蛋白在阳离子主动转运中的作用。在BR中， 有11个脯氨酸残基。其中三个(Pro-50、91和 186)被深深嵌入(x-螺旋B、C和F， 分别进行了分析。这些残基在相关的视紫红质中是保守的 源自几种细菌的菌株。守恒的存在 稳定螺旋中的脯氨酸残基通常不是预期的，并且 绕射研究表明，运动发生在这些螺旋中， 进一步表明，这些机构发挥了重要作用。通过以下方式检测 X-Pro肽键附近蛋白质骨架变化的SS核磁共振研究 Br光循环可能表明结构变化之间的相关性 已知存在于视网膜生色团中，更普遍的是 蛋白质构象的变化。关于这种相关性的信息 会让我们对质子的机制有一个更完整的了解 泵浦及其相关的视网膜结构变化 发色团和蛋白质骨架。为了检查这样的脊椎 变化，我们已经在同位素标记的BR上进行了核磁共振实验 在LA、M和M+N状态下。对于双标签[I-I 3C-X]，[ 15N-Pro]-BRS(X=Thr，Tyr，Val)， 用REDOR和CP/MAS对其15N化学位移进行了归属 三个螺旋嵌入的脯氨酸和~(13)C化学位移 继续进行X种氨基酸。在此过程中这些化学位移的变化 BR光循环反映了蛋白质构象的变化。 要区分各个组件，必须有良好的信噪比 在不同的光谱中。解决方案是另一个关键因素：它 研究表明，Pro-91在M， 状态向下移动了1.8ppm，相对于 适应光的状态。

It has been suggested that the X-proline peptide bonds play a key role in the active transport of cations by membrane proteins. In bR, there are eleven proline residues. Three of these (Pro-50, 91 and 186) are deeply embedded in the middle of (x-helices B, C, and F, respectively. These residues are conserved in related rhodopsins derived from several strains of bacteria. The presence of conserved proline residues in stable helices is not generally expected, and diffraction studies indicate that motions occur in these helices, further suggesting an important role for these. The detection by SSNMR of protein backbone changes near X-Pro peptide bonds during the bR photocycle may indicate correlations between the structural changes that are known to occur in the retinal chromophore, and more general protein conformational changes. Information about such correlations will give us a more complete picture of the mechanism of proton pumping and the related structural changes in both the retinal chromophore and protein backbone. In order to examine such backbone changes, we have performed NMR experiments on isotopically labeled bR in the LA, M. and M,,+N states. For doubly labeled [ I- I 3C-X],[ 15N-Pro]-bRs (X=Thr, Tyr, Val), the difference spectroscopy between REDOR and CP/MAS was carried out to assign the 15N chemical shifts of the three helix-embedded prolines and the 13C chemical shifts of the proceeding X amino acids. The changes in these chemical shifts during the bR photocycle reflect the changes in the protein conformation. Good signal-to-noise is mandatory to distinguish individual components in a difference spectrum. Resolution is another crucial factor: it has been indicated that the 15N chemical shift of Pro-91 in the M,, state is shifted downfield by 1.8 ppm relative to that in the light-adapted state.