Mapping the binding site of ligands to proteins using chemical exchange parameters by NMR Spectroscopy

Abstract

NMR spectroscopy, along with X-ray crystallography, have advanced to such an extent that structure aided drug design is no longer just a concept on paper. With a myriad of techniques in array, NMR spectroscopy is routinely used to screen ligands, locate binding site and design site specific molecules. Almost all NMR experiments takes advantage of the fundamental behavior of nuclei namely, the chemical exchange and relaxation phenomena, which can explain the dynamic nature of macromolecules (proteins) and their complexes (protein + ligand) in solution. Classical Bloch-McConnell equations are commonly used to study mechanisms ranging from simple to complex interactions in a quantitative way. We adopted the same approach, to understand the relationship between NMR derived kinetic parameters and the underlying interaction mechanism for protein-ligand systems. Using hBcl<SUB>XL</SUB> (protein) and BH3I-1 (ligand) as a standard system for the fast exchange regime (weak binding case), we have shown that the rate of change within a population from the free to bound state, can differentiate the binding site residues from the non binding site residues. The analysis is carried out by an in house written `c? program `Auto-FACE?, which uses a genetic algorithm to optimize kinetic (K<SUB>eq</SUB>) and spectral parameters (&#916&#969) after performing appropriate mechanism dependent free ligand corrections. Further, adopting the transition probability approach, a more comprehensive dynamic line shape analysis was automated and implemented to study different chemical exchange regimes without invoking any approximations. MCL-1 (protein) and NOXA-B (peptide), a typical slow exchange system (tight binding case), was analyzed and showed that there are regime dependent limitations on using kinetic parameters to interpret binding processes.