Structural Characterization of Protein-Ligand Complexes Using Dissolution DNP Assisted NMR Spectroscopy

Abstract

Structural information on protein-ligand complexes is of key interest in pharmaceutical research, as it provides rational guidance for optimizing ligand affinity and selectivity. An efficient tool for probing protein-ligand interactions is nuclear magnetic resonance (NMR) spectroscopy. Detailed structural information related to the binding can be obtained via the observation of polarization transfer by the nuclear Overhauser effect (NOE). In this dissertation, hyperpolarization of nuclear spins by dissolution dynamic nuclear polarization (D-DNP) is combined with both ligand-observed and protein-observed NMR to enhance the sensitivity of the NOE detection. A hyperpolarized NMR experiment is developed for detecting NOEs between two ligand spins by monitoring intra-ligand polarization transfer. The measurement of a complete intra-ligand NOE build-up curve obtained from a single hyperpolarization allows the detection of binding. Cross-relaxation rates are determined between ligand proton spins, which contain distance information. In addition to the signal enhancement provided by D-DNP, the efficiency in the intra-ligand NOE measurement is increased with protein immobilization on large bead particles. This ligand-observed DDNP approach may in the future be used for probing protein-ligand interactions in natural environments with live cells. A protein-observed NMR method based on detecting hyperpolarization transfer from ligand to protein is designed for characterizing specific intermolecular interactions. Fast acquisition of intermolecular NOEs, followed by combining the experimental data with computational docking, allows the determination of molecular structure in protein-ligand complexes. To resolve and identify specific atom-to-atom NOEs, 1D ¹H NMR spectroscopy is coupled with a ¹³C single-quantum coherence selection on the protein side and a selective inversion on ligand resonances. The resolution of NOE measurements is further improved through fast multi-dimensional NMR spectroscopy with Hadamard encoding of ligand signals. The detected intermolecular NOE contacts are used to score ligand poses generated by a docking program. A ligand structure in the active site of the protein dihydrofolate reductase is determined using experimental NOEs from a total of four DNP NMR experiments.