Nachliel Esther, Gutman Menachem, Tittor Jörg, Oesterhelt Dieter
Laser Laboratory for Fast Reactions in Biology, Department of Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel.
Biophys J. 2002 Jul;83(1):416-26. doi: 10.1016/S0006-3495(02)75179-5.
The cytoplasmic surface of the BR (initial) state of bacteriorhodopsin is characterized by a cluster of three carboxylates that function as a proton-collecting antenna. Systematic replacement of most of the surface carboxylates indicated that the cluster is made of D104, E161, and E234 (Checover, S., Y. Marantz, E. Nachliel, M. Gutman, M. Pfeiffer, J. Tittor, D. Oesterhelt, and N. Dencher. 2001. Biochemistry. 40:4281-4292), yet the BR state is a resting configuration; thus, its proton-collecting antenna can only indicate the presence of its role in the photo-intermediates where the protein is re-protonated by protons coming from the cytoplasmic matrix. In the present study we used the D96N and the triple (D96G/F171C/F219L) mutant for monitoring the proton-collecting properties of the protein in its late M state. The protein was maintained in a steady M state by continuous illumination and subjected to reversible pulse protonation caused by repeated excitation of pyranine present in the reaction mixture. The re-protonation dynamics of the pyranine anion was subjected to kinetic analysis, and the rate constants of the reaction of free protons with the surface groups and the proton exchange reactions between them were calculated. The reconstruction of the experimental signal indicated that the late M state of bacteriorhodopsin exhibits an efficient mechanism of proton delivery to the unoccupied-most basic-residue on its cytoplasmic surface (D38), which exceeds that of the BR configuration of the protein. The kinetic analysis was carried out in conjunction with the published structure of the M state (Sass, H., G. Büldt, R. Gessenich, D. Hehn, D. Neff, R. Schlesinger, J. Berendzen, and P. Ormos. 2000. Nature. 406:649-653), the model that resolves most of the cytoplasmic surface. The combination of the kinetic analysis and the structural information led to identification of two proton-conducting tracks on the protein's surface that are funneling protons to D38. One track is made of the carboxylate moieties of residues D36 and E237, while the other is made of D102 and E232. In the late M state the carboxylates of both tracks are closer to D38 than in the BR (initial) state, accounting for a more efficient proton equilibration between the bulk and the protein's proton entrance channel. The triple mutant resembles in the kinetic properties of its proton conducting surface more the BR-M state than the initial state confirming structural similarities with the BR-M state and differences to the BR initial state.
细菌视紫红质（BR，初始）状态的细胞质表面以一组三个羧酸盐为特征，它们起到质子收集天线的作用。对大多数表面羧酸盐进行系统性替换表明，该基团由D104、E161和E234组成（Checover, S., Y. Marantz, E. Nachliel, M. Gutman, M. Pfeiffer, J. Tittor, D. Oesterhelt, and N. Dencher. 2001. Biochemistry. 40:4281 - 4292），然而BR状态是一种静止构型；因此，其质子收集天线只能表明其在光中间体中的作用，在光中间体中蛋白质会被来自细胞质基质的质子重新质子化。在本研究中，我们使用D96N和三重突变体（D96G/F171C/F219L）来监测蛋白质在晚期M状态下的质子收集特性。通过持续光照使蛋白质维持在稳定的M状态，并通过反应混合物中存在的吡喃荧光素的重复激发引起可逆脉冲质子化。对吡喃荧光素阴离子的重新质子化动力学进行动力学分析，并计算游离质子与表面基团反应的速率常数以及它们之间的质子交换反应速率常数。实验信号的重建表明，细菌视紫红质的晚期M状态表现出一种高效的机制，可将质子传递到其细胞质表面未被占据的最碱性残基（D38），这一机制比蛋白质的BR构型更高效。结合已发表的M状态结构（Sass, H., G. Büldt, R. Gessenich, D. Hehn, D. Neff, R. Schlesinger, J. Berendzen, and P. Ormos. 2000. Nature. 406:649 - 653）进行动力学分析，该结构解析了大部分细胞质表面。动力学分析与结构信息的结合导致在蛋白质表面识别出两条将质子导向D38的质子传导路径。一条路径由残基D36和E237的羧酸盐部分组成，而另一条由D102和E232组成。在晚期M状态下，两条路径的羧酸盐比BR（初始）状态下更靠近D38，这使得在主体与蛋白质的质子入口通道之间的质子平衡更高效。三重突变体在其质子传导表面的动力学特性上更类似于BR - M状态而非初始状态，这证实了与BR - M状态的结构相似性以及与BR初始状态的差异。
