ИСТИНА

Studies of amide NH protection factors detected via NMR-determined measurements of hydrogen exchange in proteins can provide detailed information about fluctuations in protein structure that lead to local or subglobal unfolding of the protein. This approach has already been shown to be useful for assessing the stability of specific hydrogen bonds within a protein and for monitoring the effects of ligand binding. We have been investigating the amide H/D exchange for dihydrofolate reductase, a pharmacologically important enzyme that is the target for several clinically used antibacterial, anti-cancer and antimalarial drugs. These antifolate drugs often bind with large positive cooperativity in the presence of the coenzyme NADPH. In the case of the antibacterial drug trimethoprim, the cooperative binding has been directly implicated in the specificity of drug binding to bacterial versus mammalian forms of DHFR which determined effectiveness of the drug. The origins of cooperative ligand binding are still poorly understood. We used the measurements of NH exchange protection factors for apo DHFR and its binary and ternary complexes with TMP and coenzyme in order to explore the correlation between ligand binding cooperativity and thermodynamics of protein unfolding. Ligand binding induces long-range effect on amide H/D exchange. It was found that protection effects strongly correlate with tightness of ligand binding to DHFR. Ligand-induced protection effects of trimethoprim and co-enzyme show non-additivity similar by its magnitude to the cooperative effect of binding of these ligands. It was seen that cooperative interaction that leads to an increase of amide protections spread through the network of hydrogen bonds and polar interactions. In order to investigate the origins of such non-additivity in details, temperature effects on protection factors were examined and enthalpies and entropies of local protein unfolding processes were calculated. Obtained results indicate that conformational flexibility of protein plays an important role in molecular recognition processes and cooperative binding of ligands to biomolecule.