Rapid Quantification of Protein-Ligand Binding via F NMR Lineshape Analysis.

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.