Brown L S, Váró G, Needleman R, Lanyi J K
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
Biophys J. 1995 Nov;69(5):2103-11. doi: 10.1016/S0006-3495(95)80081-0.
The second half of the photocycle of the light-driven proton pump bacteriorhodopsin includes proton transfers between D96 and the retinal Schiff base (the M to N reaction) and between the cytoplasmic surface and D96 (decay of the N intermediate). The inhibitory effects of decreased water activity and increased hydrostatic pressure have suggested that a conformational change resulting in greater hydration of the cytoplasmic region is required for proton transfer from D96 to the Schiff base, and have raised the possibility that the reversal of this process might be required for the subsequent reprotonation of D96 from the cytoplasmic surface. Tilt of the cytoplasmic end of helix F has been suggested by electron diffraction of the M intermediate. Introduction of bulky groups, such as various maleimide labels, to engineered cysteines at the cytoplasmic ends of helices A, B, C, E, and G produce only minor perturbation of the decays of M and N, but major changes in these reactions when the label is linked to helix F. In these samples the reprotonation of the Schiff base is accelerated and the reprotonation of D96 is strongly retarded. Cross-linking with benzophenone introduced at this location, but not at the others, causes the opposite change: the reprotonation of the Schiff base is greatly slowed while the reprotonation of D96 is accelerated. We conclude that, consistent with the structure from diffraction, the proton transfers in the second half of the photocycle are facilitated by motion of the cytoplasmic end of helix F, first away from the center of the protein and then back.
光驱动质子泵细菌视紫红质光循环的后半部分包括质子在天冬氨酸96（D96）和视黄醛席夫碱之间的转移（M到N反应）以及在细胞质表面和D96之间的转移（N中间体的衰减）。水分活度降低和静水压力增加的抑制作用表明，质子从D96转移到席夫碱需要一种导致细胞质区域水合作用增强的构象变化，并且增加了一种可能性，即后续从细胞质表面使D96再质子化可能需要该过程的逆转。通过M中间体的电子衍射表明了螺旋F细胞质端的倾斜。在螺旋A、B、C、E和G的细胞质端将庞大基团（如各种马来酰亚胺标签）引入到工程化半胱氨酸中，仅对M和N的衰减产生轻微扰动，但当标签连接到螺旋F时，这些反应会发生重大变化。在这些样品中，席夫碱的再质子化加速，而D96的再质子化受到强烈抑制。在此位置引入二苯甲酮进行交联，但在其他位置不进行，会导致相反的变化：席夫碱的再质子化大大减慢，而D96的再质子化加速。我们得出结论，与衍射结构一致，光循环后半部分的质子转移通过螺旋F细胞质端的运动促进，首先远离蛋白质中心，然后再返回。
