细菌视紫红质多重突变体结构变化及其中间态的寿命

采用基因定点突变的方法, 构建了细菌视紫红质(Bacteriorhodopsin, BR)的3种突变体蛋白, 即单突变体BRE194Q、三突变体BRI119T/T121S/A126T和四突变体BRI119T/T121S/A126T/E194Q. 测定了突变体和野生型BR在水溶液和聚乙烯醇(PVA)膜中的紫外-可见吸收光谱和拉曼光谱, 采用显微视频录像技术记录了PVA膜中野生型和3个突变体样品的M态寿命. 与野生型BR相比较, 在水溶液中, 单突变体的可见吸收光谱的最大吸收峰发生了轻微红移, 三突变体和四突变体的最大吸收峰则分别发生了11.0和12.0 nm的明显蓝移. 在PVA膜中, 3个突变体BR的可见吸收光谱的最大吸收峰均发生蓝移, 四突变体BR的最大吸收峰为557 nm, 蓝移达15.0 nm. 四突变体BR在水溶液中的共振拉曼光谱不仅表现有与M态特征相关的1567和1573 cm-1谱带, 还有L态特征带1334 cm-1及N态特征带1200, 1328, 1530和1549 cm-1. 在PVA膜中的样品与在水溶液中的比较, 四突变体共振拉曼光谱的1334和1549 cm-1带消失, 同时1187 cm-1带的强度下降. 显微视频录像技术记录的PVA膜中样品的M态寿命表明, 野生型BR的M态寿命最短, 单突变体的M态寿命小于1.0 s, 三突变体的寿命为3.0 s, 四突变体的寿命为2.0 s.

Structure Change and Intermediate's Lifetime of Bacteriorhodopsin and Its Multipoint Mutants

The site-specific residue replacements E194Q, I119T/T121S/A126T and I119T/T121S /A126T/E194Q were introduced into bop gene and expressed in halobacterium salinarium L33 with the vector pXL-NovR, respectively. The photoreaction of three mutants BRE194Q, BRI119T/T121S/A126T and BRI119T/T121S/A126T/E194Q(the quadruple mutant) of bacteriorhodopsin(BR) and WT-BR(wild type BR) was investigated in solution and in PVA films at pH=7.0 by UV-Vis absorption spectrum and Resonance Raman spectra. The lifetime of M intermediate of the three mutants in PVA films were recorded by employing photomicrography. In distilled water, the visible absorption maximum of BRI119T/T121S/A126T and BRI119T/T121S/A126T/E194Q mutants were blue shifted notably by 11 and 12 nm respectively and the BRE194Q's was weakly red shifted in comparison with WT-BR. In PVA film, BRI119T/T121S/A126T/E194Q mutant caused a blue shift of the visible absorption spectrum from 568 to 557 nm. The characteristic bands of Resonance Raman spectra of BRI119T/T121S/A126T/E194Q mutant were observed at 1334cm-1 of the L intermediate, 1200, 1328, 1530 and 1549 cm-1 of the N intermediate and 1567 and 1573 cm-1 of M intermediate in solution. However, the 1334 and 1549 cm-1 bands of the quadruple mutant disappeared and the intensity of 1187 cm-1 band decreased in PVA film. The lifetime of M intermediate of BRE194Q was less than 1.0 s by photomicrography. The lifetime of M intermediates of BRI119T/T121S/A126T and BRI119T/T121S/A126T/E194Q were 3.0 s and 2.0 s, respectively.