D97N突变对光受体蛋白古紫质-4质子泵和能量转换效率的影响
发布时间：2019-01-28 22:29
【摘要】：古紫质-4(aR4)是新近发现的古生菌Halobacterium speciesxz515菌红膜上的唯一的光敏视黄醛蛋白,和细菌视紫红质(bR)具有相似的质子泵功能,但在中性pH条件下,两者的质子释放和摄取顺序相反。深入研究质子供体天冬氨酸97(Aspartic acid 97,D97)对蛋白光循环、质子泵功能和能量转化效率影响的作用机理,并从分子水平上揭示aR4质子释放和摄取时序反转内在原因具有重大的指导意义。研究工作采用基因定点突变技术,在宿主细胞Halobacterium speciesL33中成功表达出了 aR4的单突变体D97N,以及相对应的bR单突变体D96N。采用固体核磁共振技术、紫外-可见吸收光谱、闪光动力学光谱和低温透射红外光谱等谱学手段,对蛋白视黄醛键合区、光反应中间态、质子泵功能以及ATP生成率等进行了原生膜环境条件下的原位表征。闪光动力学光谱测定结果表明,D97突变可造成aR4的M态衰减时间延长至秒级,并使蛋白丧失质子泵功能,而对应的D96N突变则造成了 bR的M态衰减时间延长和O态的消失,但是蛋白仍具有质子泵功能。固体核磁共振、紫外-可见光谱、蛋白聚集状态及其ATP生成率等数据显示,不论是aR4中的D97还是bR中的D96,都距视黄醛发色团有10A之远,突变没有对视黄醛键合区产生显著的影响,但对质子泵功能和蛋白的三聚结构产生了一定的影响,使得突变体菌株ATP的生成率下降了 50%。低温透射红外光谱测定结果表明,与bR中的D85不同,aR4中的D86质子化过程提前到了L态,并一直维持到了N态。所有研究结果揭示,同为质子供体的D97和D96在aR4和bR光循环中发挥着不同的作用,表明aR4与bR存在着不同的光循环中间态,而这与二者质子释放和摄取时序反转有着直接的对应关系。
[Abstract]:Paleoviolin-4 (aR4) is the only Guang Min retinaldehyde protein on the erythrocyte membrane of Halobacterium speciesxz515, which has similar proton pump function to bacteriorhodopsin (bR), but under neutral pH condition. The order of proton release and uptake is opposite. The effects of proton donor aspartic acid 97 (Aspartic acid 97 / D97 on protein photocycling, proton pump function and energy conversion efficiency were studied. It is of great significance to reveal the internal causes of proton release and uptake time reversal of aR4 at molecular level. The single aR4 mutant D97N and the corresponding bR single mutant D96N were successfully expressed in host cell Halobacterium speciesL33 by site-directed mutation. By means of solid-state nuclear magnetic resonance spectroscopy, UV-Vis absorption spectrum, flash kinetic spectroscopy and low-temperature transmission infrared spectroscopy, the binding region of protein retinaldehyde and the intermediate state of photoreaction were studied. The proton pump function and ATP formation rate were characterized in situ under the condition of primary membrane environment. The results of flash kinetic spectroscopy show that D97 mutation can prolong the M state decay time of aR4 to the second order and make the protein lose the proton pump function. The corresponding D96N mutation results in the prolongation of M state decay time and the disappearance of O state in bR. But protein still has proton pump function. Solid nuclear magnetic resonance (NMR), UV-Vis spectroscopy, protein aggregation state and their ATP production rate showed that both D97 in aR4 and D96 in bR were 10 A away from the retinol chromophore. The mutation had no significant effect on the retinaldehyde binding region, but had a certain effect on the proton pump function and the trimeric structure of the protein, resulting in a 50% decrease in the ATP production rate of the mutant strain. The results of low temperature transmission infrared spectroscopy show that, unlike D85 in bR, the protonation of D86 in aR4 advances to L state and maintains to N state. All the results show that D97 and D96, which are the same proton donors, play different roles in the optical cycle of aR4 and bR, indicating that there are different intermediate states in the optical cycle between aR4 and bR. This has a direct relationship with proton release and uptake time reversal.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q51
本文编号：2417370
[Abstract]:Paleoviolin-4 (aR4) is the only Guang Min retinaldehyde protein on the erythrocyte membrane of Halobacterium speciesxz515, which has similar proton pump function to bacteriorhodopsin (bR), but under neutral pH condition. The order of proton release and uptake is opposite. The effects of proton donor aspartic acid 97 (Aspartic acid 97 / D97 on protein photocycling, proton pump function and energy conversion efficiency were studied. It is of great significance to reveal the internal causes of proton release and uptake time reversal of aR4 at molecular level. The single aR4 mutant D97N and the corresponding bR single mutant D96N were successfully expressed in host cell Halobacterium speciesL33 by site-directed mutation. By means of solid-state nuclear magnetic resonance spectroscopy, UV-Vis absorption spectrum, flash kinetic spectroscopy and low-temperature transmission infrared spectroscopy, the binding region of protein retinaldehyde and the intermediate state of photoreaction were studied. The proton pump function and ATP formation rate were characterized in situ under the condition of primary membrane environment. The results of flash kinetic spectroscopy show that D97 mutation can prolong the M state decay time of aR4 to the second order and make the protein lose the proton pump function. The corresponding D96N mutation results in the prolongation of M state decay time and the disappearance of O state in bR. But protein still has proton pump function. Solid nuclear magnetic resonance (NMR), UV-Vis spectroscopy, protein aggregation state and their ATP production rate showed that both D97 in aR4 and D96 in bR were 10 A away from the retinol chromophore. The mutation had no significant effect on the retinaldehyde binding region, but had a certain effect on the proton pump function and the trimeric structure of the protein, resulting in a 50% decrease in the ATP production rate of the mutant strain. The results of low temperature transmission infrared spectroscopy show that, unlike D85 in bR, the protonation of D86 in aR4 advances to L state and maintains to N state. All the results show that D97 and D96, which are the same proton donors, play different roles in the optical cycle of aR4 and bR, indicating that there are different intermediate states in the optical cycle between aR4 and bR. This has a direct relationship with proton release and uptake time reversal.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q51
【参考文献】
相关期刊论文 前2条
1 ;Primary structure of helix C to helix G of a new retinal protein in H.sp.xz515[J];Chinese Science Bulletin;2000年12期
2 李庆国,陈水月,黄静娟,徐德强,张纪忠,张燕;四株盐杆菌中类紫质分子的探测[J];生物物理学报;1993年02期
，本文编号：2417370
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