Nmr Analysis of Protein-small Molecule Interactions

INTRODUCTION Advances in NMR technology over the last 20 years have made information on solution structures of proteins increasingly available. High-resolution solution structures of proteins as large as 20-25 kDa are tractable in most cases, and recent advances allowed Kay to solve the structure of an 82 kDa protein. These advances in NMR analysis of protein structure have made possible the study of protein-ligand interactions by NMR. From NMR data, detailed solution structures of protein-ligand complexes are accessible, and multiple methods for NMR-based analysis of protein-ligand interactions have been reported. Typically, proteins are labeled with NMR-active N and/or C nuclei to enhance backbone NMR signals. In addition, H-labels are often used to attenuate proton signals of the protein. Protein NMR spectra are analyzed in the presence and absence of ligand; based on ligand-induced changes in the NMR data, information about the location of ligand binding is obtained. The dissociation constant (Kd) for the ligand of interest is calculated directly from NMR data when the rate of ligand exchange is fast with respect to the NMR timescale. For ligands with slow exchange kinetics, conventional binding assays for Kd determination are more time-efficient. CHEMICAL SHIFT MAPPING (CSM) CSM compares protein spectra in the presence and absence of ligand. Ligand binding alters the local chemical environment in the binding site, resulting in chemical shift perturbations; these perturbations are correlated, or “mapped,” to the associated protein residues to determine the binding site. The orientation of the ligand in the binding site is determined from protein-ligand NOE correlations. A typical NMR experiment for CSM of protein binding sites is N-H heteronuclear single-quantum correlation (N-HSQC), a two-dimensional experiment that correlates N nuclei with attached protons (one-bond N-H coupling). Thus, amide N-H correlations of N-labeled proteins are readily observed, and N-HSQC CSM is accomplished by correlating observed chemical shift perturbations in N-H cross-peaks to protein residues. STRUCTURE-ACTIVITY RELATIONSHIPS (SAR) BY NMR SAR by NMR is a CSM approach to ligand optimization developed by Fesik and coworkers at Abbott Laboratories. In this technique, two ligands occupying distinct, proximal sites are identified by N-HSQC CSM. Optimization of the two ligands and subsequent covalent tethering of the two “fragments” leads to a ligand which typically exhibits improved binding over either of the first two. While this approach requires the researcher to have some structural information on the target protein, it