Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
J Phys Chem B. 2010 May 6;114(17):5895-902. doi: 10.1021/jp912125z.
Fluorescence correlation spectroscopy (FCS) is a robust method for the detection of intramolecular dynamics in proteins but is also susceptible to interference from other dynamic processes such as triplet kinetics and photobleaching. We describe an approach for the detection of intramolecular dynamics in proteins labeled with a FRET dye pair based on global fitting to the two autocorrelation functions (green-green and red-red) and the two cross-correlation functions (green-red and red-green). We applied the method to detect intramolecular dynamics in the Ca(2+) signaling protein calmodulin. Dynamics were detected on the 100 mus time scale in Ca(2+)-activated calmodulin, whereas in apocalmodulin dynamics were not detected on this time scale. Control measurements on a polyproline FRET construct (Gly-Pro(15)-Cys) demonstrate the reliability of the method for isolating intramolecular dynamics from other dynamic processes on the microsecond time scale and confirm the absence of intramolecular dynamics of polyproline. We further show the sensitivity of the initial amplitudes of the FCS auto- and cross-correlation functions to the presence of multiple FRET states, static or dynamic. The FCS measurements also show that the diffusion of Ca(2+)-calmodulin is slower than that of apocalmodulin, indicating either a larger average hydrodynamic radius or shape effects resulting in a slower translational diffusion.
荧光相关光谱（FCS）是一种用于检测蛋白质内部动力学的强大方法，但也容易受到其他动态过程的干扰，如三重态动力学和光漂白。我们描述了一种基于对两个自相关函数（绿-绿和红-红）和两个互相关函数（绿-红和红-绿）的全局拟合来检测用荧光共振能量转移（FRET）染料对标记的蛋白质内部动力学的方法。我们将该方法应用于检测 Ca(2+)信号蛋白钙调蛋白的分子内动力学。在 Ca(2+)-激活的钙调蛋白中，在 100 μs 的时间尺度上检测到动力学，而在无钙调蛋白中，在该时间尺度上未检测到动力学。对聚脯氨酸 FRET 构建体（Gly-Pro(15)-Cys）的控制测量证明了该方法用于从微秒时间尺度上的其他动态过程中分离分子内动力学的可靠性，并证实了聚脯氨酸不存在分子内动力学。我们还进一步表明，FCS 自相关和互相关函数的初始幅度对存在多个 FRET 状态（静态或动态）非常敏感。FCS 测量还表明，Ca(2+)-钙调蛋白的扩散速度比无钙调蛋白慢，这表明平均水动力半径较大或形状效应导致翻译扩散速度较慢。
