INTERPRETATION OF FLUORESCENCE DECAYS IN PROTEINS USING CONTINUOUS LIFETIME DISTRIBUTIONS
INTERPRETATION OF FLUORESCENCE DECAYS IN PROTEINS USING CONTINUOUS LIFETIME DISTRIBUTIONS
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DOI：
10.1016/s0006-3495(87)83420-3
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发表时间：
1987-06-01
影响因子：
3.4
通讯作者：
PRENDERGAST, FG
中科院分区：
文献类型：
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作者：
ALCALA, JR;GRATTON, E;PRENDERGAST, FG
由于色氨酸激发态对周围环境的敏感性，蛋白质中色氨酸发射的衰减通常是复杂的。传统的使用指数成分的衰变曲线分析是基于对具有特定蛋白质构象的每个成分的识别。另一种方法假设蛋白质可以表现出大量的构象，并且在室温下，构象之间的相互转化速率可以与激发态衰变速率相同的数量级。根据这一假设，使用寿命值的连续分布来执行蛋白质发射的分析。蛋白质平均构象的数目、每个构象周围的迁移率范围以及构象之间的相互转化速率决定了寿命分布的特征。用多频相位荧光法测量了一些单一色氨酸蛋白质的荧光衰减，并用指数、单峰和双峰概率密度函数进行了分析，得到了色氨酸残基可以在单势垒中移动的模型的寿命分布的解析形式。对于核糖核酸酶T1和神经毒素变异体3，两个指数和双峰概率密度函数的和给出了类似的结果，而对于磷脂酶A2，描述衰退需要三个指数或双峰概率密度函数。研究了荧光衰减与温度的关系。研究发现，在较低的温度下，寿命分布较宽，并向较长的寿命值移动。对色氨酸在缓冲液和某些色氨酸衍生物中的衰减进行了分析，给出了单指数衰减。只有三个可调参数的单势垒寿命分布对所研究的所有情况都有很好的拟合，但在磷脂酶A2的情况下，描述单势垒分布的参数的温度依赖性表明该模型在较低的温度下是不充分的，这表明多个势垒可以更好地描述该蛋白质的衰变。
The decay of the tryptophanyl emission in proteins is often complex due to the sensitivity of the tryptophan excited state to its surroundings. The traditional analysis of the decay curve using exponential components is based on the identification of each component with a particular protein conformation. An alternative approach assumes that proteins can exhibit a large number of conformations and that, at room temperature, the interconversion rate between conformations can be of the same order of magnitude as the excited-state decay rate. Following this assumption, the analysis of the protein emission was performed using continuous distributions of lifetime values. The number of average protein conformations, the range of mobility around each conformation, and the rate of interconversion between conformations determines the characteristics of the lifetime distribution. The fluorescence decay from some single tryptophan proteins was measured using multifrequency phase fluorometry and analyzed using a sum of exponentials, unimodal and bimodal probability-density functions, and the analytical form for lifetime distribution obtained for a model in which the tryptophan residue can move in a single potential well. For ribonuclease T1 and neurotoxin variant 3, the sum of two exponentials and bimodal probability-density functions gave comparable results, whereas for phospholipase A2, the description of the decay required three exponentials or bimodal probability-density functions. Also the temperature dependence of the fluorescence decay was investigated. It was found that the lifetime distribution was broader and shifted toward longer lifetime values at lower temperature. The analysis of the decay of tryptophan in buffer and of some tryptophan derivatives gave single-exponential decays. The single-potential well lifetime distribution which has only three adjustable parameters, gave good fits for all cases investigated, but in the case of phospholipase A2 the temperature dependence of the parameters that describe the single-potential well distribution indicated the inadequacy of this model at lower temperature, suggesting that multiple potential wells can describe better the decay for this protein.