Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis

Abstract

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 19 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 19 F spectra to those from two-dimensional 1 H-15 N heteronuclear single quantum coherence spectra. Their agreement demonstrates that one-dimensional 19 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 19 F NMR lineshape analysis, and we anticipate that this analysis will be an effective tool for rapidly characterizing the energetics of protein interactions.

19F NMR as a tool in chemical biology

“…By taking a novel approach to the use of PrOF NMR, Stadmiller et al have recently developed a pioneering methodology based on 1D 19 F NMR line shape analysis that can be utilized to simultaneously determine both thermodynamic equilibrium binding constants (K) and kinetic rates of association (k on ) and dissociation (k off ) in protein-ligand binding events [37]. In order to achieve this, they analysed the binding of four different proline-rich peptides to a 5-FTrp-labelled Src homology 3 (SH3) recognition protein domain.…”

A Practical Perspective on the Roles of Solution NMR Spectroscopy in Drug Discovery

“…Lastly, 19 F-NMR can be readily utilized to rank binding affinities of different compounds, which serves a strategy to screen compounds bound to a specific site when a reference compound is available [83,125]. 19 F-NMR has recently become the most attractive tool in FBDD because more compound libraries and more sensitive probes have been developed [126,127]. A number of NMR experiments can be conducted to detect protein binding to ligands with diverse affinities (mM to nM).…”

Analysis of Conformational Exchange Processes using Methyl-TROSY-Based Hahn Echo Measurements of Quadruple-Quantum Relaxation

“…Solution NMR spectroscopy is a powerful tool for the characterization of macromolecular dynamics over a range of timescales relevant to biological function 1 : from backbone and sidechain disorder on ps-ns timescales, characterized by S 2 order parameters [2][3][4] ; rotational diffusion and domain motions, characterized by rotational correlation times, τc 5,6 ; through to real-time measurements of kinetics on timescales of seconds and beyond, following rapid mixing, temperature or pressure jumps [7][8][9] . NMR is also particularly well suited to the analysis of reversible chemical exchange on timescales of the order of microseconds to milliseconds, via lineshape analysis across a titration series [10][11][12] or using sophisticated pulse sequences such as ZZ-exchange spectroscopy, chemical exchange saturation transfer (CEST), and Carr-Purcell-Meiboom-Gill (CPMG) and R1ρ relaxation dispersion 13,14 .…”

“…Unfortunately however, the sensitivity of these experiments is somewhat low, particularly for large systems, due to rapid relaxation through C-H dipolar interactions during the constant time (CT) CPMG relaxation period. An alternative approach to the detection of chemical exchange is the analysis of the relaxation and cross-correlated relaxation of multiple quantum coherences, 12 and Δ 12 , defined as the sum and difference of ZQ and DQ relaxation rates (Fig. 1):…”