Dynamic Structural Analyzes of the Photointermediates of Bacteriorhodopsin

本研究的主要目的是对细菌视紫红质（bR）的各种光中间体进行结构分析，以了解其光驱动质子泵的分子机制。尽管D85N突变体bR不能通过光反应产生m中间体，但碱性D85N具有与m中间体相似的性质。比较了D85N在碱性pH和中性pH下的结构。为了记录不同pH下同一取向样品在同一位置的x射线衍射图，我们开发了一种铵态处理方法。从中性到碱性的衍射谱变化类似于野生型bR和D96N从大态到m -中间体的衍射谱变化。通过差分傅立叶合成，在碱性条件下，螺旋C、F和G周围发生了显著的结构变化。这些结构变化在M中间体的结构变化中是常见的。D85N/D96N的变化与D85N/D96N相似，但变化幅度远小于D85N。D85N/D96N在中性pH下的x射线衍射谱清楚地表明，即使在中性pH下，双突变体的一部分仍呈现m型结构，因此可以得出希夫碱的去质子化稳定了m型结构，而m型结构又刺激了希夫碱的去质子化。希夫碱质子只能以m型结构与细胞质表面相互作用，而质子只能以大态结构与细胞外表面相互作用。用动力学光谱和x射线衍射研究了不同pH下的m衰变过程。结构由m型向大态的逆转与席夫碱的再生密切相关。利用汞标记的半胱氨酸取代突变体bR，我们发现汞的位置可以通过x射线衍射确定。这一技术将用于进一步详细定量描述结构变化。少

The major purpose of this researchi is the structural analysis of each photointermediate of bacteriorhodopsin (bR), in order to understand the molecular mechanism of its light-driven proton pump.Despite that D85N mutant bR does not generate the M-intermediate by photo-reaction, alkaline D85N has similar properties to the M-intermediate. The structure of D85N at alkaline pH was compared with the structure at neutral pH.In order to record the X-ray diffraction patterns at the same place of the same oriented specimen under different pH,we developed an ammonium treatment method. The changes in diffraction profiles from neutral to alkaline pH resemble to those from the grand state to the M-intermediate of wild type bR and D96N.The structural changes occurred at alkaline pH are remarkable around helices C,F and G by a difference Fourier synthesis. These structural changes are common for the structural changes in the M intermediate. Although the similar changes were observed for D85N/D96N,the … More extent of the changes were much smaller than that of D85N.X-ray diffraction profile of D85N/D96N at neutral pH clearly indicates that some portion of the double mutant take the M-like structure even at neutral pH.We conclude that the deprotonation of Schiff base stabilizes the M-type structure, and that the M-type structure stimulates the deprotonation of Schiff base. Schiff base proton can interact with the cytoplasmic surface only in the M-type structure, while the proton can interact with the extracellular surface only in the grand state structure.The M-decay processes at various pH were studied by kinetic spectroscopy and kinetic X-ray diffraction. The reversion of the structure from the M-type to grand state is closely correlated with the reprotonation of Schiff base. Using mercury-labeled cysteine substitution mutant bR,we revealed that the mercury position can be identified by X-ray diffraction. This technique will be utilized for the further quantitative description of the structural change in detail. Less