Réat V, Patzelt H, Ferrand M, Pfister C, Oesterhelt D, Zaccai G
Institut de Biologie Structurale Commissariat à l'Energie Atomique-Centre National de la Recherche Scientifique, 41 Avenue des Martyrs, F-38027 Grenoble Cedex 1, France.
Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):4970-5. doi: 10.1073/pnas.95.9.4970.
We show that dynamics of specific amino acids within a protein can be characterized by neutron spectroscopy and hydrogen-deuterium labeling, and we present data on the motions of a selected set of groups within bacteriorhodopsin (BR), the retinal-based proton pump in the purple membrane of halophilic Archaea. Elastic incoherent neutron scattering experiments allow the definition of motions in the nano- to picosecond time scale and have revealed a dynamical transition from a harmonic to a softer, anharmonic atomic fluctuation regime in the global behavior of proteins. Biological activity in proteins is correlated with this transition, suggesting that flexibility is required for function. Elastic incoherent neutron scattering is dominated by H atom scattering, and to study the dynamics of a selected part of BR, fully deuterated purple membrane with BR containing H-retinal, H-tryptophan, and H-methionine was prepared biosynthetically in Halobacterium salinarum. These amino acids cluster in the functional center of the protein. In contrast to the protein globally, the thermal motions of the labeled atoms were found to be shielded from solvent melting effects at 260 K. Above this temperature, the labeled groups appear as more rigid than the rest of the protein, with a significantly smaller mean square amplitude of motion. These experimental results quantify the dynamical heterogeneity of BR (which meets the functional requirements of global flexibility), on the one hand, to allow large conformational changes in the molecule and of a more rigid region in the protein, on the other, to control stereo-specific selection of retinal conformations.
我们表明，蛋白质中特定氨基酸的动力学可以通过中子光谱学和氢氘标记来表征，并且我们展示了嗜盐古菌紫膜中基于视黄醛的质子泵细菌视紫红质（BR）内一组选定基团运动的数据。弹性非相干中子散射实验能够定义纳秒到皮秒时间尺度内的运动，并揭示了蛋白质整体行为中从谐波到更软的非谐波原子涨落状态的动态转变。蛋白质中的生物活性与这种转变相关，这表明功能需要灵活性。弹性非相干中子散射以氢原子散射为主，为了研究BR选定部分的动力学，在盐生盐杆菌中通过生物合成制备了含有H - 视黄醛、H - 色氨酸和H - 甲硫氨酸的完全氘代紫膜与BR。这些氨基酸聚集在蛋白质的功能中心。与蛋白质整体情况相反，发现标记原子的热运动在260 K时免受溶剂熔化效应的影响。高于此温度，标记基团显得比蛋白质的其余部分更刚性，其运动的均方振幅明显更小。这些实验结果一方面量化了BR的动态异质性（满足全局灵活性的功能要求），以允许分子发生大的构象变化，另一方面量化了蛋白质中更刚性区域的动态异质性，以控制视黄醛构象的立体特异性选择。
