Stadmiller Samantha S, Aguilar Jhoan S, Waudby Christopher A, Pielak Gary J
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina.
Department of Structural and Molecular Biology, University College London, London, United Kingdom.
Biophys J. 2020 May 19;118(10):2537-2548. doi: 10.1016/j.bpj.2020.03.031. Epub 2020 Apr 15.
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-dimensional F 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-dimensional F spectra to those from two-dimensional H-N heteronuclear single quantum coherence spectra. Their agreement demonstrates that one-dimensional F 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 of F NMR lineshape analysis, and we anticipate that this analysis will be an effective tool for rapidly characterizing the energetics of protein interactions.
氟的掺入非常适合许多核磁共振技术，并且将氟掺入蛋白质和用于药物发现的片段库中已变得越来越普遍。在这里，我们使用一维氟核磁共振线形分析来量化氟标记的Src同源3（SH3）蛋白结构域与四种富含脯氨酸的肽结合的动力学和平衡热力学。SH3结构域是最大且特征最明确的蛋白质识别结构域家族之一，在真核细胞信号传导中具有多种功能。首先，我们使用酰胺质子温度系数表明，氟掺入SH3对游离态和结合态仅引起微小的结构变化。然后，我们将一维氟谱的线形分析结果与二维氢 - 氮异核单量子相干谱的结果进行比较。它们的一致性表明，一维氟线形分析是一种强大、低成本且快速的替代传统基于异核单量子相干实验的方法。数据表明结合受扩散限制，并表明过渡态与游离态高度相似。我们还测量了结合与温度的函数关系。在平衡时，结合由焓驱动，并且源于缔合的高度正的活化焓以及小的熵贡献。我们的结果与使用不同技术的研究结果一致，为氟核磁共振线形分析的实用性提供了额外证据，并且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。
