Tanio M, Tuzi S, Yamaguchi S, Kawaminami R, Naito A, Needleman R, Lanyi J K, Saitô H
Department of Life Science, Himeji Institute of Technology, Harima Science Garden City, Kuoto 3-chome, Kamigori, Hyogo, 678-1297, Japan.
Biophys J. 1999 Sep;77(3):1577-84. doi: 10.1016/S0006-3495(99)77005-0.
We have recorded (13)C NMR spectra of [3-(13)C]Ala-labeled wild-type bacteriorhodopsin (bR) and its mutants at Arg(82), Asp(85), Glu(194), and Glu(204) along the extracellular proton transfer chain. The upfield and downfield displacements of the single carbon signals of Ala(196) (in the F-G loop) and Ala(126) (at the extracellular end of helix D), respectively, revealed conformational differences in E194D, E194Q, and E204Q from the wild type. The same kind of conformational change at Ala(126) was noted also in the Y83F mutant, which lacks the van der Waals contact between Tyr(83) and Ala(126) present in the wild type. The absence of a negative charge at Asp(85) in the site-directed mutant D85N induced global conformational changes, as manifested in displacements or suppression of peaks from the transmembrane helices, cytoplasmic loops, etc., as well as the local changes at Ala(126) and Ala(196) seen in the other mutants. Unexpectedly, no conformational change at Ala(126) was observed in R82Q (even though Asp(85) is protonated at pH 6) or in D85N/R82Q. The changes induced in the Ala(126) signal when Asp(85) is uncharged could be interpreted therefore in terms of displacement of the positive charge of Arg(82) toward Tyr(83), where Ala(126) is located. It is possible that disruption of the proton transfer chain after protonation of Asp(85) in the photocycle could cause the same kind of conformational change we detect at Ala(196) and Ala(126). If so, the latter change would be also the result of rearrangement of the side chain of Arg(82).
我们记录了沿细胞外质子转移链在精氨酸（82）、天冬氨酸（85）、谷氨酸（194）和谷氨酸（204）位点进行[3 - （13）C]丙氨酸标记的野生型细菌视紫红质（bR）及其突变体的（13）C NMR谱。丙氨酸（196）（在F - G环中）和丙氨酸（126）（在螺旋D的细胞外端）单个碳信号的高场和低场位移分别揭示了E194D、E194Q和E204Q与野生型之间的构象差异。在Y83F突变体中也注意到了丙氨酸（126）处的相同类型构象变化，该突变体缺乏野生型中存在的酪氨酸（83）和丙氨酸（126）之间的范德华接触。定点突变体D85N中天冬氨酸（85）处负电荷的缺失诱导了全局构象变化，表现为跨膜螺旋、细胞质环等的峰位移或抑制，以及其他突变体中在丙氨酸（126）和丙氨酸（196）处的局部变化。出乎意料的是，在R82Q（即使在pH 6时天冬氨酸（85）被质子化）或D85N/R82Q中未观察到丙氨酸（126）处的构象变化。因此，当Asp（85）不带电荷时在丙氨酸（126）信号中诱导的变化可以根据精氨酸（82）的正电荷向丙氨酸（126）所在的酪氨酸（83）的位移来解释。有可能在光循环中天冬氨酸（85）质子化后质子转移链的破坏会导致我们在丙氨酸（196）和丙氨酸（126）处检测到的相同类型的构象变化。如果是这样，后者的变化也将是精氨酸（82）侧链重排的结果。
