Estimating protein-protein interaction affinity in living cells using quantitative Forster resonance energy transfer measurements
Estimating protein-protein interaction affinity in living cells using quantitative Forster resonance energy transfer measurements
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DOI：
10.1117/1.2799171
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发表时间：
2007-09-01
影响因子：
3.5
通讯作者：
Ikeda, Stephen R.
中科院分区：
文献类型：
--
作者：
Chen, Huanmian;Puhl, Henry L., III;Ikeda, Stephen R.
我们以前已经证明，福斯特共振能量转移（FRET）的效率和供体和受体荧光团的相对浓度可以确定在活细胞中使用三立方宽场荧光显微镜。在这里，我们扩展的方法来估计有效的平衡解离常数（Kd）和固有的FRET效率（E-max）的相互作用的供体-受体对。假设双分子相互作用，预测的FRET效率是供体浓度，受体浓度，Kd和Emax的函数。我们估计Kd和Emax的预测和测量的FRET效率之间的平方误差（SSE）的总和最小化。这是通过检查假设Kd和Emax值的矩阵的SSE值的拓扑来实现的。应用F检验，计算Kd和E-max的95%置信度等值线。我们通过在HeLa细胞中表达由FKBP 12-Cerulean和Frb-Venus组成的诱导型FRET融合对来测试该方法。由于FKBP 12-雷帕霉素和Frb的Kd已经通过分析测定，因此可以用基于蛋白质浓度的值校准相对Kd（以荧光单位计）。所描述的方法应该是有用的比较在活细胞中的蛋白质-蛋白质相互作用的亲和力。(C)2007年，由光学仪器工程师协会（Society of Photo-Optical Instrumentation Engineers）主办。
We have previously demonstrated that Forster resonance energy transfer (FRET) efficiency and the relative concentration of donor and acceptor fluorophores can be determined in living cells using three-cube wide-field fluorescence microscopy. Here, we extend the methodology to estimate the effective equilibrium dissociation constant (Kd) and the intrinsic FRET efficiency (E-max) of an interacting donor-acceptor pair. Assuming bimolecular interaction, the predicted FRET efficiency is a function of donor concentration, acceptor concentration, Kd, and Emax. We estimate Kd and Emax by minimizing the sum of the squared error (SSE) between the predicted and measured FRET efficiency. This is accomplished by examining the topology of SSE values for a matrix of hypothetical Kd and Emax values. Applying an F-test, the 95% confidence contour of Kd and E-max is calculated. We test the method by expressing an inducible FRET fusion pair consisting of FKBP12-Cerulean and Frb-Venus in HeLa cells. As the Kd for FKBP12-rapamycin and Frb has been analytically determined, the relative Kd (in fluorescence units) could be calibrated with a value based on protein concentration. The described methodology should be useful for comparing protein-protein interaction affinities in living cells. (C) 2007 Society of Photo-Optical Instrumentation Engineers.