Protein Oligomerization Equilibria and Kinetics Investigated by Fluorescence Correlation Spectroscopy: A Mathematical Treatment
Protein Oligomerization Equilibria and Kinetics Investigated by Fluorescence Correlation Spectroscopy: A Mathematical Treatment
复制标题
DOI：
10.1021/jp507741r
复制
发表时间：
2014-10-30
影响因子：
3.3
通讯作者：
Levitus, Marcia
中科院分区：
文献类型：
--
作者：
Kanno, David M.;Levitus, Marcia
荧光相关光谱 (FCS) 是一种越来越多地用于研究蛋白质寡聚平衡和动力学的技术。每个单独的 FCS 衰减都以其幅度和特征扩散时间为特征，两者都对蛋白质的解离程度敏感。在这里，我们提供了一种数学处理，将这些可观测值与感兴趣的参数联系起来：不同蛋白质解离步骤的平衡常数及其相应的解离和缔合动力学速率常数。我们重点关注两种最常见的蛋白质同源寡聚物类型（二聚体和四聚体）以及与实验设计相关的实验变量（蛋白质浓度、标记蛋白质的分数浓度）。对具有不同解离常数的蛋白质的理论预期的分析是实验设计和数据分析的一个关键方面，如果不对系统进行物理上的精确处理就无法进行。特别是，我们表明使用一些常用的经验模型对FCS数据进行分析可能会导致对实验结果的严重误解。
Fluorescence correlation spectroscopy (FCS) is a technique that is increasingly being used to investigate protein oligomerization equilibria and dynamics. Each individual FCS decay is characterized by its amplitude and a characteristic diffusion time, both of which are sensitive to the degree of dissociation of the protein. Here, we provide a mathematical treatment that relates these observables with the parameters of interest: the equilibrium constants of the different protein dissociation steps and their corresponding dissociation and association kinetic rate constants. We focused on the two most common types of protein homooligomers (dimers and tetramers) and on the experimental variables relevant for the design of the experiment (protein concentration, fractional concentration of labeled protein). The analysis of the theoretical expectations for proteins with different dissociation constants is a key aspect of experiment design and data analysis and cannot be performed without a physically accurate treatment of the system. In particular, we show that the analysis of FCS data using some commonly used empirical models may result in a serious misinterpretation of the experimental results.