Yamaguchi S, Tuzi S, Yonebayashi K, Naito A, Needleman R, Lanyi J K, Saitô H
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Kouto 3-chome, Kamigori, Hyogo 678-1297, Japan.
J Biochem. 2001 Mar;129(3):373-82. doi: 10.1093/oxfordjournals.jbchem.a002867.
We have recorded (13)C NMR spectra of [2-(13)C]-, [1-(13)C]-, [3-(13)C],- and [1,2,3-(13)C(3)]Ala-labeled bacteriorhodopsin (bR), and its mutants, A196G, A160G, and A103C, by means of cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques, to reveal the conformation and dynamics of bR, with emphasis on the loop and C-terminus structures. The (13)C NMR signals of the loop (C-D, E-F, and F-G) regions were almost completely suppressed from [2-(13)C]-, [1-(13)C]Ala-, and [1-(13)C]Gly-labeled bR, due to the presence of conformational fluctuation with correlation times of 10(-4) s that interfered with the peak-narrowing by magic angle spinning. The observation of such suppressed peaks for specific residues provides a unique means of detecting intermediate frequency motions on the time scale of ms or micros in the surface loops of membrane proteins. Instead, the three well-resolved (13)C CP-MAS NMR signals of [2-(13)C]Ala-bR, at 50.38, 49.90, and 47.96 ppm, were ascribed to the C-terminal alpha-helix previously proposed from the data for [3-(13)C]Ala-bR: the former two peaks were assigned to Ala 232 and 238, in view of the results of successive proteolysis experiments, while the highest-field peak was ascribed to Ala 235 prior to Pro 236. Even such (13)C NMR signals were substantially broadened when (13)C NMR spectra of fully labeled [1,2,3-(13)C]Ala-bR were recorded, because the broadening and splitting of peaks due to the accelerated transverse relaxation rate caused by the increased number of relaxation pathways through a number of (13)C-(13)C homo-nuclear dipolar interactions and scalar J couplings, respectively, are dominant among (13)C-labeled nuclei. In addition, approximate correlation times for local conformational fluctuations of different domains, including the C-terminal tail, C-terminal alpha-helix, loops, and transmembrane alpha-helices, were estimated by measurement of the spin-lattice relaxation times in the laboratory frame and spin-spin relaxation times under the conditions of cross-polarization-magic angle spinning, and comparative study of suppressed specific peaks between the CP-MAS and DD-MAS experiments.
我们通过交叉极化 - 魔角旋转（CP - MAS）和偶极去耦 - 魔角旋转（DD - MAS）技术记录了[2 - (¹³)C] -、[1 - (¹³)C] -、[3 - (¹³)C] - 和[1,2,3 - (¹³)C₃]丙氨酸标记的细菌视紫红质（bR）及其突变体A196G、A160G和A103C的(¹³)C NMR谱，以揭示bR的构象和动力学，重点关注环和C末端结构。由于存在相关时间为10⁻⁴ s的构象波动，干扰了魔角旋转导致的峰变窄，[2 - (¹³)C] -、[1 - (¹³)C]丙氨酸 - 和[1 - (¹³)C]甘氨酸标记的bR中环（C - D、E - F和F - G）区域的(¹³)C NMR信号几乎完全被抑制。对于特定残基观察到这种被抑制的峰，提供了一种独特的方法来检测膜蛋白表面环中毫秒或微秒时间尺度上的中频运动。相反，[2 - (¹³)C]丙氨酸 - bR在50.38、49.90和47.96 ppm处的三个分辨良好的(¹³)C CP - MAS NMR信号归因于先前从[3 - (¹³)C]丙氨酸 - bR数据中提出的C末端α - 螺旋：鉴于连续蛋白酶解实验的结果，前两个峰被指定为Ala 232和238，而最高场峰在Pro 236之前归因于Ala 235。当记录完全标记的[1,2,3 - (¹³)C]丙氨酸 - bR的(¹³)C NMR谱时，即使是这样的(¹³)C NMR信号也会显著变宽，因为分别通过大量(¹³)C - (¹³)C同核偶极相互作用和标量J耦合导致的横向弛豫速率加快引起的峰变宽和分裂，在(¹³)C标记的核中占主导地位。此外，通过测量实验室坐标系中的自旋 - 晶格弛豫时间以及交叉极化 - 魔角旋转条件下的自旋 - 自旋弛豫时间，并对CP - MAS和DD - MAS实验之间被抑制的特定峰进行比较研究，估计了不同结构域（包括C末端尾巴、C末端α - 螺旋、环和跨膜α - 螺旋）局部构象波动的近似相关时间。
