一种近红外荧光蛋白PAiRFP1及其突变体V276G的光活化效率研究

摘要

PAiRFP1 is one Near-infrared fluorescent protein modified from Agp2,one bacteriophytochrome from Agrobacterium tumefaciens ,using protein engineering strategy.Being different from other bacteriophytochrome-based fluorescent protein, PAiRFP1 exhibited specific photoactivatable behavior.This property favored the enhancement of signal-to-noise ratio when PAiRFP1 was used as fluorescence probe in vivo imaging.Little useful information has been available concerning what factors would affect the photoactivation behavior of PAiRFP1.We herein investigated the effect of protein concentration,pH,metal ion and redox condition on the photoactivation of PAiRFP1.It was found that (1)there is no linear relationship between the maxi-mum photoactivation efficiency and the protein concentration.(2 )The maximum photoactivation efficiency increased from 36.8％ to 52.0％ and 60.8％ upon increasing pH from 6.5 to 7.8 and 9.0.(3)The metal ions exerted no significant effect on the maximum photoactivation efficiency of PAiRFP1.Similar phenomenon was observed following the treatment of hydrogen peroxide or dithiothreitol.Apart from these factors,one mutant named V276G was constructed,where the valine residue at site 276 was mutated to glycine residue.This displacement resulted in one decrease of the maximum photoactivation efficiency from～50.0％ to 19.4％.Additionally,when DTT was used to the V276G mutant,the maximum photoactivation efficiency was ob-served to increase from 19.4％ to 27.1％.In this paper,we also studied the changes of photoactivation rate induced by these factors.These results will provide useful information and guidance for the application of PAiRFP1 into in-vivo imaging.%PAiRFP1是一种以根癌农杆菌中细菌光敏色素Agp2为基础,经过蛋白工程手段改造后获得的近红外荧光蛋白.与其他近红外荧光蛋白不同,PAiRFP1具有光活化行为.这一特性可以有效改善近红外荧光蛋白在体内的成像信噪比.然而,关于光活化行为的影响因素却研究甚少.主要采用荧光光谱学手段研究了PAiRFP1蛋白浓度、pH、金属离子、氧化还原环境对于PAiRFP1光活化行为的影响,结果发现:(1)PAiRFP1的最大光活化效率与该近红外荧光蛋白的浓度不呈线性关系;(2)随着pH从6.5升高至7.8和9.0,PAiRFP1的最大光活化效率从36.8％提高至52.0％和60.8％;(3)金属离子K+,Na+,Ca2+的加入对于PAiRFP1的最大光活化效率没有显著影响;类似现象在H 2 O2和DTT的处理的情况下也被观察到.除了上述影响因素外,还发现当PAiRFP1中276位的缬氨酸被突变为甘氨酸产生V276G突变体后,蛋白的最大光活化效率从～50.0％下降至19.4％.DTT处理后导致V276G突变体最大光活化效率从19.4％提高到了27.1％.此外,比较研究了各种影响因素对于光活化速率的影响.以上结果为今后更好地将此类光激活的近红外蛋白应用于活体成像中提供了更好地理论指导和优化解决方案.