Kawase Y, Tanio M, Kira A, Yamaguchi S, Tuzi S, Naito A, Kataoka M, Lanyi J K, Needleman R, Saitô H
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Kouto 3-chome, Kamigori, Hyogo, Japan.
Biochemistry. 2000 Nov 28;39(47):14472-80. doi: 10.1021/bi0015820.
According to previous X-ray diffraction studies, the D85N mutant of bacteriorhodopsin (bR) with unprotonated Schiff base assumes a protein conformation similar to that in the M photointermediate. We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled D85N and D85N/D96N mutants at ambient temperature to examine how conformation and dynamics of the protein backbone are altered when the Schiff base is protonated (at pH 7) and unprotonated (at pH 10). Most notably, we found that the peak intensities of three to four [3-(13)C]Ala-labeled residues from the transmembrane alpha-helices, including Ala 39, 51, and 53 (helix B) and 215 (helix G), were suppressed in D85N and D85N/D96N both from CP-MAS (cross polarization-magic angle spinning) and DD-MAS (dipolar decoupled-magic angle spinning) spectra, irrespective of the pH. This is due to conformational change and subsequent acquisition of intermediate time-range motions, with correlation times in the order of 10(-)(5) or 10(-)(4) s, which interferes with proton decoupling frequency or frequency of magic angle spinning, respectively, essential for an attempted peak-narrowing to achieve high-resolution NMR signals. Greater changes were achieved, however, at pH 10, which indicate large-amplitude motions of transmembrane helices upon deprotonation of Schiff base and the formation of the M-like state in the absence of illumination. The spectra detected more rapid motions in the extracellular and/or cytoplasmic loops, with correlation times increasing from 10(-)(4) to 10(-)(5) s. Conformational changes in the transmembrane helices were located at helices B, G, and D as viewed from the above-mentioned spectral changes, as well as at 1-(13)C-labeled Val 49 (helix B), 69 (B-C loop), and [3-(13)C]Ala-labeled Ala 126 (D-helix) signals, in addition to the cytoplasmic and extracellular loops. Further, we found that in the M-like state the charged state of Asp 96 at the cytoplasmic side substantially modulated the conformation and dynamics of the extracellular region through long-distance interaction.
根据先前的X射线衍射研究，具有未质子化席夫碱的细菌视紫红质（bR）的D85N突变体呈现出与M光中间体相似的蛋白质构象。我们在室温下记录了[3-(13)C]丙氨酸和[1-(13)C]缬氨酸标记的D85N和D85N/D96N突变体的(13)C NMR谱，以研究当席夫碱质子化（pH 7）和未质子化（pH 10）时蛋白质主链的构象和动力学是如何改变的。最值得注意的是，我们发现，来自跨膜α-螺旋的三到四个[3-(13)C]丙氨酸标记残基的峰强度，包括丙氨酸39、51和53（螺旋B）以及215（螺旋G），在D85N和D85N/D96N的CP-MAS（交叉极化-魔角旋转）和DD-MAS（偶极去耦-魔角旋转）谱中均被抑制，与pH无关。这是由于构象变化以及随后获得的中间时间范围运动，其相关时间约为10^(-5)或10^(-4)秒，这分别干扰了质子去耦频率或魔角旋转频率，而这对于尝试使峰变窄以获得高分辨率NMR信号至关重要。然而，在pH 10时实现了更大的变化，这表明席夫碱去质子化时跨膜螺旋的大幅度运动以及在无光照情况下形成类似M的状态。光谱检测到细胞外和/或细胞质环中的运动更快，相关时间从10^(-4)增加到10^(-5)秒。从上述光谱变化来看，跨膜螺旋的构象变化位于螺旋B、G和D，以及1-(13)C标记的缬氨酸49（螺旋B）、69（B-C环）和[3-(13)C]丙氨酸标记的丙氨酸126（D-螺旋）信号处，以及细胞质和细胞外环。此外，我们发现，在类似M的状态下，细胞质侧的天冬氨酸96的带电状态通过长距离相互作用显著调节了细胞外区域的构象和动力学。
