Kinsey R A, Kintanar A, Tsai M D, Smith R L, Janes N, Oldfield E
J Biol Chem. 1981 May 10;256(9):4146-9.
We have obtained the first deuterium NMR spectra of an individual membrane protein, bacteriorhodopsin in the purple membrane of Halobacterium halobium R1. Biosynthetic isotopic enrichment with [gamma-2H6]valine and high field Fourier transform operation permitted rapid data acquisition on intact membranes, including measurement of relaxation times. At some temperatures high quality spectra could be obtained in less than 1 s. [U-14C]Valine tracer studies indicate that less than or equal to 2% of valine added to the growth medium is broken down and incorporated into other membrane constituents. The NMR results indicate that the valine side chain is a rather rigid structure. Motion about C alpha-C beta is slow (less than 10(5) s-1) at growth temperature, While motion about C beta-C gamma is as expected fast (much greater than 10(5) s-1) at all accessible temperatures. The activation energy for methyl group rotation from spin-lattice relaxation data between -75 and 53 degrees C is approximately 2.4 kcal/mol, in good agreement with previous 1H NMR studies on solid alkanes. Preliminary data on [gamma-2H6]valine-labeled Acholeplasma laidlawii B (PG9) cell membranes are also presented. Our results strongly suggest that it should now be possible to observe in great detail the motions of any type of amino acid side chain in membrane proteins, including the effects of lipid composition on protein dynamics.
我们获得了单个膜蛋白——嗜盐菌R1紫膜中的细菌视紫红质的首个氘核磁共振谱。用[γ-2H6]缬氨酸进行生物合成同位素富集以及高场傅里叶变换操作，使得能够在完整膜上快速采集数据，包括弛豫时间的测量。在某些温度下，不到1秒就能获得高质量的谱图。[U-14C]缬氨酸示踪研究表明，添加到生长培养基中的缬氨酸分解并掺入其他膜成分的比例小于或等于2%。核磁共振结果表明缬氨酸侧链是一种相当刚性的结构。在生长温度下，围绕Cα-Cβ的运动很慢（小于10^5 s-1），而在所有可达到的温度下，围绕Cβ-Cγ的运动如预期那样很快（远大于10^5 s-1）。根据-75至53摄氏度之间的自旋晶格弛豫数据，甲基旋转的活化能约为2.4千卡/摩尔，这与先前对固态烷烃的1H核磁共振研究结果非常吻合。还展示了[γ-2H6]缬氨酸标记的莱氏无胆甾原体B（PG9）细胞膜的初步数据。我们的结果有力地表明，现在应该能够非常详细地观察膜蛋白中任何类型氨基酸侧链的运动，包括脂质组成对蛋白质动力学的影响。
