Size-Dependent Relationships between Protein Stability and Thermal Unfolding Temperature Have Important Implications for Analysis of Protein Energetics and High-Throughput Assays of Protein–Ligand Interactions
Size-Dependent Relationships between Protein Stability and Thermal Unfolding Temperature Have Important Implications for Analysis of Protein Energetics and High-Throughput Assays of Protein–Ligand Interactions
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蛋白质稳定性和热解折叠温度之间的尺寸依赖性关系对于蛋白质能量学分析和蛋白质与配体相互作用的高通量测定具有重要意义
DOI：
10.1021/acs.jpcb.7b05684
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Raleigh, Daniel P.
中科院分区：
文献类型：
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作者：
Watson, Matthew D.;Monroe, Jeremy;Raleigh, Daniel P.
蛋白质稳定性的变化通常报告为解链温度 ΔTM 的变化，或特定温度下展开自由能 ΔΔG° 的变化。使用来自 16 种蛋白质的 866 个突变体的数据，我们检查了 ΔΔG° 和 ΔTM 之间的关系。每种蛋白质都观察到线性关系。不同蛋白质的 ΔTM 与 ΔΔG° 绘图的斜率为 N–1，其中 N 是蛋白质中残基的数量。因此，相对于大蛋白质观察到的效果，给定的 ΔG° 变化会导致小蛋白质的 TM 发生更大的变化。分析表明，可以通过内插 TM 的测量值来获得突变体 ΔΔG° 的合理估计。 ΔΔG°和ΔTM之间的关系对于蛋白质-配体结合的高通量测定的设计和解释具有影响。所谓的热位移测定依赖于配体与折叠状态结合所导致的稳定性的增加。得出的定量关系表明观察到的热位移 ΔTM 的尺度为 N–1。因此，热位移测定对于配体与较大蛋白质的结合相当不敏感。
Changes in protein stability are commonly reported as changes in the melting temperature, ΔTM, or as changes in unfolding free energy at a particular temperature, ΔΔG°. Using data for 866 mutants from 16 proteins, we examine the relationship between ΔΔG°and ΔTM. A linear relationship is observed for each protein. The slopes of the plots of ΔTMvs ΔΔG°for different proteins scale asN–1, whereNis the number of residues in the protein. Thus, a given change in ΔG°causes a much larger change inTMfor a small protein relative to the effect observed for a large protein. The analysis suggests that reasonable estimates of ΔΔG°for a mutant can be obtained by interpolating measured values ofTM. The relationship between ΔΔG°and ΔTMhas implications for the design and interpretation of high-throughput assays of protein–ligand binding. So-called thermal shift assays rely upon the increase in stability which results from ligand binding to the folded state. Quantitative relationships are derived which show that the observed thermal shift, ΔTM, scales asN–1. Hence, thermal shift assays are considerably less sensitive for ligand binding to larger proteins.