Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes
Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes
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DOI：
10.1021/bi101414z
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发表时间：
2010-12-28
期刊：
影响因子：
2.9
通讯作者：
Hellinga, Homme W.
中科院分区：
文献类型：
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作者：
Layton, Curtis J.;Hellinga, Homme W.
蛋白质配体相互作用的定量对于系统生物学、药物发现和生物工程是必不可少的。配体诱导的蛋白质热稳定性的变化提供了结合的一般的、可量化的特征，并且可以用染料如Sypro橙子（SO）监测，其在与未折叠的蛋白质相互作用时增加其荧光发射强度。这种方法是一种实验简单，经济，高通量的方法，观察热熔体使用常用的实时聚合酶链反应仪器。然而，定量分析需要仔细考虑染料介导的报告机制和基础的热力学模型。我们通过分析配体介导的解链温度中点值的变化来确定亲和力常数。在配体滴定系列中，根据在共同参考温度下稳定性自由能的变化确定配体亲和力。热力学参数是通过将报告热变性的实验信号的逆一阶导数与包含线性或非线性基线模型的方程拟合来获得的。我们应用这些方法，以适应蛋白质熔体监测与SO表现出突出的非线性后过渡基线。SO可以扰乱它所报告的均衡。我们分析的情况下，配体结合到原生和变性状态或原生状态，只有蛋白质：配体的化学计量需要明确治疗的情况下。
The quantification of protein ligand interactions is essential for systems biology, drug discovery, and bioengineering. Ligand-induced changes in protein thermal stability provide a general, quantifiable signature of binding and may be monitored with dyes such as Sypro Orange (SO), which increase their fluorescence emission intensities upon interaction with the unfolded protein. This method is an experimentally straightforward, economical, and high-throughput approach for observing thermal melts using commonly available real-time polymerase chain reaction instrumentation. However, quantitative analysis requires careful consideration of the dye-mediated reporting mechanism and the underlying thermodynamic model. We determine affinity constants by analysis of ligand-mediated shifts in melting-temperature midpoint values. Ligand affinity is determined in a ligand titration series from shifts in free energies of stability at a common reference temperature. Thermodynamic parameters are obtained by fitting the inverse first derivative of the experimental signal reporting on thermal denaturation with equations that incorporate linear or nonlinear baseline models. We apply these methods to fit protein melts monitored with SO that exhibit prominent nonlinear post-transition baselines. SO can perturb the equilibria on which it is reporting. We analyze cases in which the ligand binds to both the native and denatured state or to the native state only and cases in which protein:ligand stoichiometry needs to treated explicitly.