Kinsey R A, Kintanar A, Oldfield E
J Biol Chem. 1981 Sep 10;256(17):9028-36.
We have obtained the first deuterium NMR spectra of individual types of aromatic amino acids in a defined membrane protein, bacteriorhodopsin, in the photosynthetic purple membrane of Halobacterium halobium R1. Isotopic labeling and high field (8.5 Tesla) operation permitted relatively rapid data acquisition at a variety of temperatures. At the temperature of growth (37 degrees C), we find that all 7 tryptophan residues are rigid on the time scale of the NMR experiment (approximately 10(-5) s), except for likely librational motions of approximately 10 degrees amplitude. By contrast, nearly all (9 +/- 2) of the 11 tyrosines and (13 +/- 2) 13 phenylalanines undergo rapid (greater than 10(5) s-1) 2-fold rotational flips about C gamma-C zeta, causing formation of line shapes dominated by effectively axially asymmetric (asymmetry parameter eta = 0.66) deuteron electric field gradient tensors. On cooling the phenylalanine- and tyrosine-labeled samples to approximately -30 degrees C, all such motions are frozen out, i.e. occur at rates less than 10(4) s-1, and axially symmetric (eta approximately 0.05) line shapes are observed. At T greater than 91 degrees C, phenylalanine-, tyrosine-, and tryptophan-labeled membrane spectra undergo dramatic narrowing to an isotropic line of approximately 4-9 kHz width. This transition is a reflection of the loss of tertiary structure in the membrane protein with resultant fast unrestricted motion of these aromatic side chains, and is only partly reversible. These results, in conjunction with those obtained using [gamma-2H6]valine-labeled bacteriorhodopsin (Kinsey, R. A., Kintanar, A., Tsai, M-D., Smith, R. L., Janes, N., and Oldfield, E. (1981) J. Biol. Chem., 256, 4146-4149) indicate the rather rigid nature of amino acid side chains in the H. halobium purple membrane, the principal fast lage amplitude motions being methyl group rotation and discontinuous benzene ring "flipping."
我们已获得嗜盐栖热菌R1光合紫色膜中特定膜蛋白细菌视紫红质内各类型芳香族氨基酸的首个氘核磁共振谱。同位素标记和高场（8.5特斯拉）操作使得能在多种温度下相对快速地采集数据。在生长温度（37℃）下，我们发现所有7个色氨酸残基在核磁共振实验的时间尺度（约10⁻⁵秒）上是刚性的，除了可能存在幅度约为10度的摆动运动。相比之下，11个酪氨酸中的几乎所有（9±2个）以及13个苯丙氨酸中的（13±2个）都围绕Cγ - Cζ进行快速（大于10⁵秒⁻¹）的双折旋转翻转，导致形成主要由有效轴向不对称（不对称参数η = 0.66）的氘核电场梯度张量主导的线形。将苯丙氨酸和酪氨酸标记的样品冷却至约 - 30℃时，所有这些运动都被冻结，即发生速率小于10⁴秒⁻¹，并且观察到轴向对称（η约为0.05）的线形。在T大于91℃时，苯丙氨酸、酪氨酸和色氨酸标记的膜谱经历显著变窄，变为宽度约为4 - 9千赫兹的各向同性谱线。这种转变反映了膜蛋白三级结构的丧失以及这些芳香族侧链随之产生的快速无限制运动，并且只是部分可逆。这些结果与使用[γ - 2H₆]缬氨酸标记的细菌视紫红质所获得的结果（Kinsey, R. A., Kintanar, A., Tsai, M - D., Smith, R. L., Janes, N., and Oldfield, E. (1981) J. Biol. Chem., 256, 4146 - 4149）相结合，表明嗜盐栖热菌紫色膜中氨基酸侧链具有相当刚性的性质，主要的快速大幅度运动是甲基旋转和不连续的苯环“翻转”。
