Signal transduction via membrane protein complex : photo-signal transduction of Halobacterium salinarum

许多生物学功能由膜蛋白复合物执行，其中一个例子是盐细菌的光接收/趋光性。在这项研究中，我们研究了通过信号复合物的pharaonis phoborhodopsin（ppR，或pharaonis sensory rhodopsin）和它的转换器（pHtrII）在natronomonaspharaonis光接收。结果表明：1.ppR是一种视网膜膜蛋白。近年来，许多文献报道了视网膜蛋白在许多微生物中的存在，这些视网膜蛋白被称为微生物视紫红质。视网膜席夫碱附近的精氨酸残基在这些视紫红质中是保守的。对ppR的精氨酸残基进行了突变并研究了其作用。2.光照引起ppR的F-螺旋和氢键网络的构象变化。3.固态NMR也观察了构象变化。4.光照引起ppR的质子释放/吸收，用透明SnO_2 5.显示了pHtrII的胞质部分与ppR环之间的相互作用以及膜内的相互作用。7.光照条件下，ppR单独存在时，可将质子从内向外转运，当与pHtrll结合时，其泵送活性丧失。盐视紫红质（hR）是一种内向光驱动的Cl ~-泵，当hR被改造为能够与pHtrII结合时，复合物失去了泵活性，而突变体单独保留了泵活性。8.用等温滴定量热法测定了hR与Cl ~-结合的热力学参数。

Many biological functions are performed by membrane protein complexes, one example of which is the photo-reception/taxis of halobacterium. In this study, we investigated the photo-reception via the signaling complex of pharaonis phoborhodopsin (ppR, or pharaonis sensory rhodopsin) and its transducer (pHtrII) in Natronomonas pharaonis. Results obtained are :1.ppR is a retinal membrane protein. Recently many papers report the existence of retinal proteins in many micro-organisms, and these retinal proteins are called microbial rhodopsin. Arginine residues near the retinal Schiff base are conserved among these rhodopsins. The arginine residue in ppR was mutated and its role was investigated.2.Illumination to ppR induced conformational changes of the F-helix and hydrogen bonding networks.3.The conformational changes were also viewed by a solid-state NMR.4.Illumination elicited a proton release/uptake of ppR, which was monitored by a transparent SnO_2 electrode.5.Interaction between a cytoplasmic part of pHtrII and a loop of ppR as well as interaction within the membrane were shown.6.The intra-membranous three hydrogen bonds were verified using various mutants.7.On illumination, ppR alone can transport protons from inside to outside, and its pumping activity loses when it combined with pHtrll. Halorhodopsin (hR) is inwardly light-driven Cl^- pump, and when hR was engineered so as to be able to bind with pHtrII, the complex lost the pumping activity while the mutant alone retained the activity.8.The thermodynamic parameters of Cl- binding of hR were determined by isothermal titration calorimetry.