Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis.
Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis.
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DOI：
10.1016/j.bpj.2020.03.031
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发表时间：
2020-04
影响因子：
3.4
通讯作者：
Samantha S. Stadmiller;Jhoan S. Aguilar;C. Waudby;G. Pielak
中科院分区：
文献类型：
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作者：
Samantha S. Stadmiller;Jhoan S. Aguilar;C. Waudby;G. Pielak
氟掺入非常适合于许多NMR技术，并且将氟掺入蛋白质和片段库中用于药物发现已经变得越来越普遍。在这里，我们使用一维19 F NMR线形分析，以量化的动力学和平衡热力学的结合的氟标记的Src同源3（SH 3）蛋白质结构域的四个脯氨酸丰富的肽。SH 3结构域是蛋白质识别结构域中最大和最充分表征的家族之一，并且在真核细胞信号传导中具有多种功能。首先，我们表明，氟纳入SH 3的原因只有轻微的结构变化的自由和束缚态使用酰胺质子温度系数。然后，我们比较了一维19 F谱的线形分析结果和二维1H-15 N异质单量子相干谱的线形分析结果。他们的一致性表明，一维19 F线形分析是传统的异质单量子相干实验的一种稳健，低成本和快速的替代方案。数据表明，结合是扩散限制，并表明过渡态是高度相似的自由状态。我们还测量了结合作为温度的函数。在平衡状态下，结合是由热力学驱动的，并且产生于与小熵贡献相关联的高度正的活化焓。我们的结果与使用不同技术的研究结果一致，为19 F NMR线形分析的实用性提供了额外的证据，我们预计这种分析将成为快速表征蛋白质相互作用能量学的有效工具。
Fluorine incorporation is ideally suited to many NMR techniques, and incorporation of fluorine into proteins and fragment libraries for drug discovery has become increasingly common. Here, we use one-dimensional19F NMR lineshape analysis to quantify the kinetics and equilibrium thermodynamics for the binding of a fluorine-labeled Src homology 3 (SH3) protein domain to four proline-rich peptides. SH3 domains are one of the largest and most well-characterized families of protein recognition domains and have a multitude of functions in eukaryotic cell signaling. First, we showe that fluorine incorporation into SH3 causes only minor structural changes to both the free and bound states using amide proton temperature coefficients. We then compare the results from lineshape analysis of one-dimensional19F spectra to those from two-dimensional1H-15N heteronuclear single quantum coherence spectra. Their agreement demonstrates that one-dimensional19F lineshape analysis is a robust, low-cost, and fast alternative to traditional heteronuclear single quantum coherence-based experiments. The data show that binding is diffusion limited and indicate that the transition state is highly similar to the free state. We also measured binding as a function of temperature. At equilibrium, binding is enthalpically driven and arises from a highly positive activation enthalpy for association with small entropic contributions. Our results agree with those from studies using different techniques, providing additional evidence for the utility of19F NMR lineshape analysis, and we anticipate that this analysis will be an effective tool for rapidly characterizing the energetics of protein interactions.