The angles between the C(1)-, C(5)-, and C(9)-methyl bonds of the retinylidene chromophore and the membrane normal increase in the M intermediate of bacteriorhodopsin: direct determination with solid-state (2)H NMR.
The angles between the C(1)-, C(5)-, and C(9)-methyl bonds of the retinylidene chromophore and the membrane normal increase in the M intermediate of bacteriorhodopsin: direct determination with solid-state (2)H NMR.
复制标题
细菌视紫红质 M 中间体中视黄基发色团的 C(1)-、C(5)- 和 C(9)- 甲基键与膜正常之间的角度增加：用固态 (2)H 直接测定
作者：
Moltke,S;Wallat,I;Sakai,N;Nakanishi,K;Brown,MF;Heyn,MP
用固体氘核磁共振（~ 2 H NMR）研究了细菌视紫红质的视黄基发色团的三个甲基键在M光中间体中的取向。在这个关键中间体中，发色团具有13-顺式，15-反式构象和去质子化的Schiff碱。含有野生型或突变型D96 A细菌视紫红质的紫色膜用β-紫罗酮环的C1或C5或多烯链的C9碳的甲基中特异性氘代的视黄酸再生。通过在86%相对湿度下干燥玻璃板上的浓缩悬浮液形成取向的水合膜。在野生型样品中，通过在pH 9.5下使用盐酸胍溶液，以及在D96 A样品中，通过提高pH值，M态的寿命增加。在−50 °C下，对暗适应基态（13-顺式、15-顺式和反式、15-反式发色团的2：1混合物）、低温捕获的光适应态（全反式、15-反式）和低温捕获的M中间体（13-顺式、15-反式）进行了2 H NMR实验。细菌视紫红质首先在+5 °C的水合膜的稳定光照下完全转化为M，然后在黑暗中迅速冷却至−50 °C。从磁场中取向样品的倾斜序列和2 H NMR谱线形状的分析中，即使在存在相当程度的取向无序的情况下，也可以确定单个C-CD 3键与膜法线之间的角度。虽然在暗适应状态和光适应状态之间只检测到微小的差异，但在M状态中所有三个角度都增加。这与多烯链的C5− C13部分向细胞质表面的向上运动或增加的扭转应变一致。C 9 − CD 3键在M中显示出最大的7°取向变化。结合口袋中发色团的这种重新取向为先前的建议（基于光谱证据）提供了直接的结构支持，即螺旋F中C9−甲基和Trp 182之间的空间相互作用。
The orientations of three methyl bonds of the retinylidene chromophore of bacteriorhodopsin were investigated in the M photointermediate using deuterium solid-state NMR (2H NMR). In this key intermediate, the chromophore has a 13-cis, 15-anticonformation and a deprotonated Schiff base. Purple membranes containing wild-type or mutant D96A bacteriorhodopsin were regenerated with retinals specifically deuterated in the methyl groups of either carbon C1or C5of the β-ionone ring or carbon C9of the polyene chain. Oriented hydrated films were formed by drying concentrated suspensions on glass plates at 86% relative humidity. The lifetime of the M state was increased in the wild-type samples by applying a guanidine hydrochloride solution at pH 9.5 and in the D96A sample by raising the pH.2H NMR experiments were performed on the dark-adapted ground state (a 2:1 mixture of 13-cis, 15-synandall-trans, 15-antichromophores), the cryotrapped light-adapted state (all-trans, 15-anti), and the cryotrapped M intermediate (13-cis, 15-anti) at −50 °C. Bacteriorhodopsin was first completely converted to M under steady illumination of the hydrated films at +5 °C and then rapidly cooled to −50 °C in the dark. From a tilt series of the oriented sample in the magnetic field and an analysis of the2H NMR line shapes, the angles between the individual C−CD3bonds and the membrane normal could be determined even in the presence of a substantial degree of orientational disorder. While only minor differences were detected between dark- and light-adapted states, all three angles increase in the M state. This is consistent with an upward movement of the C5−C13part of the polyene chain toward the cytoplasmic surface or with increased torsional strain. The C9−CD3bond shows the largest orientational change of 7° in M. This reorientation of the chromophore in the binding pocket provides direct structural support for previous suggestions (based on spectroscopic evidence) for a steric interaction in M between the C9−methyl group and Trp 182 in helix F.