Yonebayashi Koka, Yamaguchi Satoru, Tuzi Satoru, Saitô Hazime
Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Garden City, Kouto 3-chome, Kamigori, Hyogo 678-1297, Japan.
Eur Biophys J. 2003 Mar;32(1):1-11. doi: 10.1007/s00249-002-0260-0. Epub 2002 Nov 1.
We have examined how cytoplasmic surface structures of [3-(13)C]Ala-labeled bacteriorhodopsin (bR), consisting of the C-terminal alpha-helix and cytoplasmic loops, are altered by site-directed mutations at the former (R227Q) and the latter (A160G, E166G, and A168G) and by cation binding, by means of displacements of the (13)C NMR peaks of Ala228 and Ala233 (C-terminal alpha-helix), Ala103 (C-D loop), and Ala160 (E-F loop). Cytoplasmic ends of the B and F helices were found to undergo fluctuation motions on the order of 10(-5) s, when such surface structures were disrupted, as viewed from suppressed (13)C NMR signals. This happens also for deionized blue membranes of wild type and A160G, with accelerated fluctuations in the loops. Further, cytoplasmic surface structures of Na(+)-regenerated purple membrane from the blue membrane were significantly modified by Ca(2+) ions up to 1 mM under relatively low ionic strength of 10 mM NaCl, although they are very similar at high ionic strength (100 mM NaCl). To interpret these findings, the following two surface structures were proposed. The C-terminal alpha-helix of the wild type at ambient temperature is involved in a perturbed type, probably tilted toward the direction of the B and F helices, to prevent unnecessary fluctuations of these helices for efficient proton uptake during the photocycle. An unperturbed type of helix is achieved when such a surface structure was disrupted at low temperature or in an M-like state. This view is consistent with previously published data for the "proton binding cluster" consisting of Asp104, Glu166, and Glu234.
我们通过观察丙氨酸228、丙氨酸233（C端α螺旋）、丙氨酸103（C-D环）和丙氨酸160（E-F环）的¹³C NMR峰的位移，研究了由C端α螺旋和细胞质环组成的[³-(¹³)C]丙氨酸标记的细菌视紫红质（bR）的细胞质表面结构是如何因前者（R227Q）和后者（A160G、E166G和A168G）的定点突变以及阳离子结合而改变的。从¹³C NMR信号被抑制的情况来看，当这些表面结构被破坏时，发现B和F螺旋的细胞质末端会发生大约10⁻⁵秒量级的波动运动。野生型和A160G的去离子化蓝色膜也会出现这种情况，环中的波动会加速。此外，在10 mM NaCl的相对低离子强度下，蓝色膜中Na⁺再生的紫色膜的细胞质表面结构会被高达1 mM的Ca²⁺离子显著改变，尽管它们在高离子强度（100 mM NaCl）下非常相似。为了解释这些发现，提出了以下两种表面结构。在环境温度下，野生型的C端α螺旋参与一种受扰类型，可能向B和F螺旋的方向倾斜，以防止这些螺旋在光循环期间进行不必要的波动，从而实现有效的质子摄取。当这种表面结构在低温或M样状态下被破坏时，会形成一种未受扰的螺旋类型。这一观点与先前发表的关于由天冬氨酸104、谷氨酸166和谷氨酸234组成的“质子结合簇”的数据一致。
