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首页> 外文期刊>The journal of physical chemistry, B. Condensed matter, materials, surfaces, interfaces & biophysical >Detecting Intramolecular Dynamics and Multiple Forster Resonance Energy Transfer States by Fluorescence Correlation Spectroscopy
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Detecting Intramolecular Dynamics and Multiple Forster Resonance Energy Transfer States by Fluorescence Correlation Spectroscopy

机译：通过荧光相关光谱法检测分子内动力学和多个Forster共振能量转移状态

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摘要

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 ah 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 μs 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.
机译：荧光相关光谱法（FCS）是检测蛋白质分子内动力学的可靠方法，但也容易受到其他动力学过程（例如三重态动力学和光漂白）的干扰。我们描述了一种基于FRET染料对标记的蛋白质中分子内动力学检测的方法，该方法基于对两个自相关函数（绿色-绿色和红色-红色）和两个互相关函数（绿色-红色和红色-红色）的全局拟合绿色）。我们应用该方法来检测Ca〜（2+）信号蛋白钙调蛋白中的分子内动力学。在Ca〜（2+）激活的钙调蛋白中，在100μs时间尺度上检测到动力学，而在载脂蛋白调节剂中，在该时间尺度上未检测到动力学。在多脯氨酸FRET构建体（Gly-Pro_（15）-Cys）上的对照测量结果证明了在微秒级时从其他动态过程中分离分子内动力学的方法的可靠性，并证实了不存在聚脯氨酸的分子内动力学。我们进一步展示了FCS自相关函数和互相关函数的初始幅度对存在多个静态或动态FRET状态的敏感性。 FCS测量还表明，Ca〜（2 +）-钙调蛋白的扩散比脱辅基钙调蛋白的扩散慢，这表明较大的平均流体动力学半径或形状效应导致较慢的平移扩散。

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