D38R突变对细菌视紫红质光循环后半程M态和N态的影响

用重叠PCR的方法，定点突变细菌视紫红质（bacteriorhodopsin，BR），克隆到单突变体BRD96N和双突变体BRD38R/D96N的基因，同源转化盐沼盐杆菌（Halobacterium salinarium）L33（BR），表达突变蛋白BRD96N和BRD38R／D96N通过显微视频方法对突变BR的M态光致变色特性进行记录，BRD38R／D96N的M态寿命为5s，约为BRD96N（M态寿命为3s）的1．7倍．对突变体的激光共聚焦拉曼光谱测定发现，在BRD96N中，1170cm^-1下移到1171cm^-1，1258cm^-1上移到1255cm^-1，而BRD38R/D96N中1170cm^-1下移到1173cm^-1；1258cm^-1。上移到1252cm^-1，而且BRD38R/D96N与BRD96N相比，1186cm^-1处的峰明显增强．近紫外区圆二色谱显示，两种突变体近紫外区的正负带虽然没有太大差别，但288nm和290nm处的极峰却差别显著．所有这些研究结果表明，细菌视紫红质Asp38（D38）突变为Arg（R）可以延长其光循环后半程M态和N态的寿命．

Effection of D38R on the Second Half of the Bacteriorhodopsin Photocycle

Bacteriorhodopsin mutagenes of D96N and D38R/D96N were cloned by the Overlapping PCR method.The mutagenes BR_(D96N) and BR_(D38R/D96N) were transformed to Halobacterium salinarium L33(BR~-) and the mutant BR_(D96N) and BR_(D38R/D96N) proteins were successfully expressed.The purified purple membrane of mutants and wild type were recorded by microvideo and analyzed with the Raman spectroscopy and the Circular Dichroism spectroscopy.The result of microvideo illustrated that the intermediate M-decay time 5 seconds of BR_(D38R/D96N) was about 1.7-fold more than the time 3 seconds of mutant BR_(D96N).In the case of the BR_(D38R/D96N),the accumulation of an N-like 13-cis species was indicated by the increased intensity of the 1 186 cm~(1)band in Raman spectroscopy.Furthermore,their CD spectrums near UV were obviously difference in pole peaks of 270-290 cm~(-1).All these results showed that the mutant D38R of BR increased the intermediate M-decay and N-decay time,which would be of the theoretical and applied value.