Ulrich A S, Watts A, Wallat I, Heyn M P
Department of Biochemistry, University of Oxford, U.K.
Biochemistry. 1994 May 10;33(18):5370-5. doi: 10.1021/bi00184a003.
Structural details about the geometry of the retinal chromophore in the binding pocket of bacteriorhodopsin are revealed by measuring the orientations of its individual methyl groups. Solid-state 2H-NMR measurements were performed on macroscopically oriented samples of purple membrane patches, containing retinal specifically deuterium-labeled at one of the three methyl groups along the polyene chain (C18, C19, C20). The deuterium quadrupole splitting of each "zero-tilt" spectrum is used to calculate the orientation of the corresponding C-CD3 bond vector with respect to the membrane normal; however, two possible solutions may arise. These ambiguities in angle could be resolved by recording a tilt series of spectra at different sample inclinations to the magnetic field and analyzing the resulting complex line shapes with the aid of computer simulations. The angles for the C18, C19, and C20 group are found to be 37 +/- 1 degree, 40 +/- 1 degree, and 32 +/- 1 degree, respectively. These highly accurate values imply that the polyene chain of the retinal chromophore is not straight but rather has an in-plane curvature and possibly an out-of-plane twist. Together with the angles of the remaining methyl groups on the cyclohexene ring that have been measured previously, an overall picture has thus emerged of the intramolecular conformation and the three-dimensional orientation of retinal within bacteriorhodopsin. The deduced geometry confirms and refines the known structural information on the chromophore, suggesting that this 2H-NMR strategy may serve as a valuable tool for other membrane proteins.
通过测量细菌视紫红质结合口袋中视网膜发色团各个甲基的取向，揭示了其几何结构细节。对紫色膜片的宏观取向样品进行了固态2H-NMR测量，这些样品中的视网膜在多烯链上的三个甲基之一（C18、C19、C20）处进行了特异性氘标记。每个“零倾斜”光谱的氘四极分裂用于计算相应的C-CD3键向量相对于膜法线的取向；然而，可能会出现两种可能的解决方案。通过在不同的样品与磁场倾斜角度下记录一系列倾斜光谱，并借助计算机模拟分析由此产生的复杂线形，可以解决角度的这些模糊性。发现C18、C19和C20基团的角度分别为37±1度、40±1度和32±1度。这些高精度值表明，视网膜发色团的多烯链不是直的，而是具有面内曲率，并且可能具有面外扭曲。结合先前测量的环己烯环上其余甲基的角度，从而得出了细菌视紫红质内视网膜的分子内构象和三维取向的整体图像。推导的几何结构证实并完善了关于发色团的已知结构信息，表明这种2H-NMR策略可能成为其他膜蛋白的有价值工具。
