葡萄球菌核酸酶蛋白的时间分辨荧光与热力学特性

摘要

葡萄球菌核酸酶(SNase)是一种小型球状蛋白,其变体常用来研究蛋白质的折叠过程.不同于之前报道的研究方法和技术手段,采用时间相关单光子计数(TCSPC)及飞秒荧光上转换技术,结合紫外吸收谱和稳态荧光光谱,研究了SNase蛋白变体 Δ+PHS和 Δ+PHS+ I92A的荧光动力学,以及不同温度下蛋白结构与热稳定性的关系,证明蛋白质内色氨酸残基可作为一种内源性探针对蛋白变体的结构折叠和热稳定性进行印证和研究.衰减相关光谱(DAS)表明了两种变体随温度变化的不同趋势,在此基础上进一步分析了这两种变体的结构折叠及热稳定性的差异.皮秒时间分辨发射光谱(T RES)显示色氨酸残基存在0.5 ns的连续光谱弛豫过程,而光谱移动量可作为SNase变体蛋白结构紧密程度的判断依据.飞秒上转换数据分析结果中,0.5 ps的DAS在光谱蓝端为正、红端为负,表明了色氨酸残基受到弛豫效应的影响.200 ps的寿命则说明色氨酸残基与周围猝灭基团之间存在电子转移过程.时间分辨荧光各向异性(anisotropy)的分析结果则说明了色氨酸残基在蛋白质体系内具有独立的局部运动,且其强弱与变体的热稳定性和热运动的整体效果有关.测量和分析色氨酸残基的时间分辨荧光性质为深入研究SNase蛋白的结构和功能提供了新的思路.%Staphylococcal nuclease is a small globular protein,whose variants are widely used in the researches on protein fold-ing.Different from methods and techniques reported in published papers,fluorescence dynamics of tryptophan residues in two staphylococcal nuclease(SNase)variants Δ+PHS and Δ+ PHS+I92A were investigated by time-correlated single photon count-ing(TCSPC)and femtosecond fluorescence upconversion techniques,combined with UV absorption and steady-state fluores-cence spectroscopy.Based on the analysis,structures and thermal stabilities of the two SNase variants were studied.The results proved that tryptophan could be used as an endogenous probe for the structural folding and thermal stability of the SNase vari-ants.Decay associated spectra(DAS)of SNase variants showed different changing trends upon temperatures.According to this, structural folding and thermal stability of the two variants were analyzed.Time-resolved emission spectra(TRES)demonstrated the 0.5 ns continuous spectral relaxation process of tryptophan residue,in which the spectral shift showed the compactness difference of folding structures of the two SNase variants.In femtosecond upconversion results,DAS of 0.5 ps lifetime had"positive blue edge and negative red edge",which showed relaxation effects on tryptophan residues in SNase variants.Moreo-ver,the lifetime of 200 ps indicated the electron transfer between tryptophan residues and surrounding quenching group.Analy-sis of time-resolved anisotropy showed that the tryptophan residues had independent segmental motion in the protein system,and its intensity was related to the thermal stability of SNase variants and the overall effect of thermal motion.Time-resolved fluo-rescence measurement and analysis of tryptophan residue helped to investigate the relationship between structure and function of protein.