Chen Huanmian, Puhl Henry L, Ikeda Stephen R
National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism, Laboratory of Molecular Physiology, Section on Transmitter Signaling, Bethesda, Maryland 20892-9411, USA.
J Biomed Opt. 2007 Sep-Oct;12(5):054011. doi: 10.1117/1.2799171.
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 (Emax) 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 Emax 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.
我们之前已经证明，使用三立方宽场荧光显微镜可以在活细胞中测定福斯特共振能量转移（FRET）效率以及供体和受体荧光团的相对浓度。在此，我们扩展该方法以估计相互作用的供体 - 受体对的有效平衡解离常数（Kd）和固有FRET效率（Emax）。假设为双分子相互作用，预测的FRET效率是供体浓度、受体浓度、Kd和Emax的函数。我们通过最小化预测和测量的FRET效率之间的平方误差总和（SSE）来估计Kd和Emax。这是通过检查假设的Kd和Emax值矩阵的SSE值拓扑来实现的。应用F检验，计算Kd和Emax的95%置信轮廓。我们通过在HeLa细胞中表达由FKBP12 - 天蓝蛋白和Frb - 维纳斯组成的可诱导FRET融合对来测试该方法。由于已经通过分析确定了FKBP12 - 雷帕霉素和Frb的Kd，相对Kd（以荧光单位计）可以用基于蛋白质浓度的值进行校准。所描述的方法对于比较活细胞中的蛋白质 - 蛋白质相互作用亲和力应该是有用的。
