BACTERIORHODOPSIN CHROMOPHORE STRUCTURE BY DEUTERIUM NMR

拟议研究的目的是用2H-核磁共振研究 细菌视紫红质的主要成分--发色团的结构 这个光驱动的质子泵。质子转移反应的速率为 严重依赖于质子给体的距离和键角 接受者。质子化的希夫碱是其中质子的来源 泵和所获得的结构信息将在 开发其机理的详细模型。每一块的角度 将发色团的五个甲基与 多烯链和β-紫罗兰酮环将根据 胎膜正常。这些角度将高精度地从 取向单轴样品~2H-核磁共振谱的四极分裂 有选择地用相应的紫膜再生 髓核化视网膜。实验将在最初的黑暗中进行 状态以及关键的光循环中间体M、N和O中，其中 发色团具有13-顺式构型或具有去质子化的希夫 基地。因此，角度的变化将提供关于 这些功能中发色团的曲率、扭曲和倾斜 重要的中间人。外部参数(pH、离子强度和 温度)和突变体将允许捕获各种中间体。 M中间体是质子转移的关键中间体 循环，并将特别注意其作为一个 再质子化开关。互补自旋-晶格弛豫实验 将执行以了解不同部分的流动性 不同中间体中的生色团。这一信息将是 对了解其他医学上重要的离子转移有价值 膜蛋白，对其结构知之甚少。 此外，细菌视紫红质还是一个重要的家族的原型。 具有七个跨膜α-螺旋的受体蛋白，包括 视觉色素视紫红质。视紫红质的激活涉及结构 它的M-II中间体的变化源于生色团和 传播到细胞质环。对类比现象的考察 BR中间体中的步骤预计将有助于 视紫红质在视觉信号中的激活机制 转导。

The objective of the proposed research is to investigate by 2H-NMR the structure of the chromophore of bacteriorhodopsin, the key component of this light-driven proton pump. The rate of proton transfer reactions is critically dependent on the distances and bond angles of proton donors and acceptors. The protonated Schiff base is the source of protons in this pump and the structural information obtained will be essential in developing detailed models of its mechanism. The angles of each of the bonds connecting the five methyl groups of the chromophore with the polyene chain and beta-ionone ring will be determined with respect to the membrane normal. These angles will be obtained with high accuracy from the quadrupole splittings of the 2H-NMR spectra of oriented uniaxial samples of purple membranes regenerated with the corresponding selectively deuterated retinals. Experiments will be carried out in the dark initial state as well as in the key photocycle intermediates M, N and O, in which the chromophore is in a 13-cis configuration or has a deprotonated Schiff base. The changes in angle will thus provide structural information about curvature, twists and tilt of the chromophore in these functionally important intermediates. External parameters (pH, ionic strength and temperature) and mutants will allow trapping of the various intermediates. The M intermediate is the key intermediate in the proton translocation cycle, and particular attention will be paid to its potential role as a reprotonation switch. Complementary spin-lattice relaxation experiments will be performed to learn about the mobility of various parts of the chromophore in the different intermediates. This information will be valuable in understanding other medically important ion translocating membrane proteins, for which much less is known about the structure. Bacteriorhodopsin is moreover the prototype of the important family of receptor proteins with seven transmembrane alpha-helices, which includes the visual pigment rhodopsin. Activation of rhodopsin involves structural changes in its M-II intermediate which originates in the chromophore and propagate to the cytoplasmic loops. An investigation of the analogous steps in the M intermediate of bR is expected to contribute to an understanding of the mechanism of activation of rhodopsin in visual signal transduction.