Y186F突变对光受体蛋白古紫质-4质子传输和能量转换的影响

发布时间：2018-07-29 07:14

【摘要】：光驱动质子泵是一类以视黄醛为主要发色团的七-跨膜光敏受体蛋白,其功能是利用质子由胞内输送到胞外所形成的氢离子浓度梯度,通过ATP的合成将光能转化为化学能。本研究以细菌古紫质-4(archaerhodopsin-4,a R4)为研究对象,旨在探究位于视黄醛结合口袋的芳香性残基酪氨酸186(Y186)对其质子泵功能以及能量转换效率的影响。利用重叠延伸PCR技术构建Y186F~L33-a R4突变体,并通过紫外-可见光吸收光谱、动力学瞬态吸收光谱以及ATP生成率测定等手段,对RC~L33-a R4(recombinant a R4)和Y186F~L33-a R4突变体进行对比研究。研究表明,相较于RC~L33-a R4,Y186F~L33-a R4突变体的紫外-可见光谱最大吸收峰发生了4 nm的蓝移,M中间态寿命延长了约5倍,O中间态寿命则延长了约2倍,同时突变之后造成了质子泵功能的明显减弱。此外,相较于重组RC~L33-a R4菌株,Y186F~L33-a R4突变体菌株的ATP生成率约降低了36.5%。因此可以判定,位于视黄醛结合口袋的Y186是一个对质子传输和能量转换起重要作用的芳香性残基
[Abstract]:Photo-driven proton pump is a kind of seven-transmembrane Guang Min receptor protein with retinoic aldehyde as the main chromophore. Its function is to convert light energy into chemical energy through the synthesis of ATP using hydrogen ion concentration gradient formed by proton transport from intracellular to extracellular. The aim of this study was to investigate the effects of aromatic residue tyrosine 186 (Y186) on the proton pump function and energy conversion efficiency of aracerhodopsin-4 (Archaerhodopin-4). Y186F~L33-a R4 mutants were constructed by overlapping extended PCR technique. The RC~L33-a R4 (recombinant a R4) and Y186F~L33-a R4 mutants were compared by UV-Vis absorption spectra, kinetic transient absorption spectra and ATP formation rate. The results show that the intermediate lifetime of blue shift M is about 5 times longer than that of RC~L33-a R _ 4 Y186FN L33-a R _ 4 mutants, but it is about 2 times longer than that of RC~L33-a R _ 4 Y186F _ (33-a) R _ 4 mutants, and the maximum absorption peak of UV-Vis spectrum occurs at 4 nm, and the intermediate lifetime of blue shift M increases by about 5 times. At the same time, the proton pump function was obviously weakened after the mutation. In addition, the ATP production rate of the recombinant RC~L33-a R4 mutant Y186FN L33-a R4 was about 36.5% lower than that of the recombinant strain Y186FN L33-a R4. Thus, it can be determined that the Y186 located in the retinaldehyde binding pocket is an aromatic residue that plays an important role in proton transport and energy conversion.
【作者单位】：华东师范大学物理与材料科学学院上海市磁共振重点实验室;
【基金】：国家自然科学基金项目(21475045)资助
【分类号】：Q78

【相似文献】