Chromophore orientation in bacteriorhodopsin determined from the angular dependence of deuterium nuclear magnetic resonance spectra of oriented purple membranes.
Chromophore orientation in bacteriorhodopsin determined from the angular dependence of deuterium nuclear magnetic resonance spectra of oriented purple membranes.
复制标题
细菌视紫红质中的发色团方向是根据定向紫色膜的氘核磁共振光谱的角度依赖性确定的。
DOI：
10.1021/bi980676v
复制
发表时间：
1998
期刊：
影响因子：
2.9
通讯作者：
Brown,MF
中科院分区：
文献类型：
--
作者：
Moltke,S;Nevzorov,AA;Sakai,N;Wallat,I;Job,C;Nakanishi,K;Heyn,MP;Brown,MF
膜蛋白中辅基的取向对于理解它们在能量转换、信号转导和离子转运中的功能作用是相当重要的。本文用~ 2 H NMR研究了细菌视紫红质（bR）的视黄基发色团的取向。用全反式-视紫红质在环己烯环C1上的一个偕甲基上立体特异性氘代再生细菌视紫红质。通过在载玻片上形成紫色膜的水合层状膜，制备了一种高度取向的样品，这是从2 H NMR中获得单个键取向所必需的。由于键角的分布和膜的取向无序性，发展了一种蒙特卡罗方法来精确模拟2 HNMR谱线的形状。通过独立测量本征线宽（1.6 kHz，来自T2 e实验）和有效四极耦合常数（38.8 - 39.8 kHz，来自粉末型样品的线形分析），减少了线形模拟中的自由参数数量。C1−（1 R）-1-CD 3键和紫膜法线之间的角度是通过同时分析20 ° C和−50 °C下磁场中单轴取向样品在不同倾角下记录的一系列2 H NMR光谱来高精度确定的。暗适应bR中的68.7 ± 2.0°的值与先前确定的C5− CD 3键的角度一起用于计算膜中环己烯环的可能取向。从2 H NMR得到的解决方案，然后结合额外的限制，从线性二色性和电子低温显微镜，以获得允许的取向的非中心对称膜结构中的视网膜。综合数据表明，多烯链上的甲基基团指向膜的细胞质侧，席夫碱的N-H键指向细胞外侧，即，朝向泵路径中质子释放的一侧。
The orientation of prosthetic groups in membrane proteins is of considerable importance in understanding their functional role in energy conversion, signal transduction, and ion transport. In this work, the orientation of the retinylidene chromophore of bacteriorhodopsin (bR) was investigated using2H NMR spectroscopy. Bacteriorhodopsin was regenerated with all-trans-retinal stereospecifically deuterated in one of the geminal methyl groups on C1of the cyclohexene ring. A highly oriented sample, which is needed to obtain individual bond orientations from2H NMR, was prepared by forming hydrated lamellar films of purple membranes on glass slides. A Monte Carlo method was developed to accurately simulate the2H NMR line shape due to the distribution of bond angles and the orientational disorder of the membranes. The number of free parameters in the line shape simulation was reduced by independent measurements of the intrinsic line width (1.6 kHz fromT2eexperiments) and the effective quadrupolar coupling constant (38.8−39.8 kHz from analysis of the line shape of a powder-type sample). The angle between the C1−(1R)-1-CD3bond and the purple membrane normal was determined with high accuracy from the simultaneous analysis of a series of2H NMR spectra recorded at different inclinations of the uniaxially oriented sample in the magnetic field at 20 and −50 °C. The value of 68.7 ± 2.0° in dark-adapted bR was used, together with the previously determined angle of the C5−CD3bond, to calculate the possible orientations of the cyclohexene ring in the membrane. The solutions obtained from2H NMR were then combined with additional constraints from linear dichroism and electron cryomicroscopy to obtain the allowed orientations of retinal in the noncentrosymmetric membrane structure. The combined data indicate that the methyl groups on the polyene chain point toward the cytoplasmic side of the membrane and the N−H bond of the Schiff base to the extracellular side, i.e., toward the side of proton release in the pump pathway.