The goal of this proposal is to investigate the role of dynamics in the long-range coupling of allosteric sites in proteins. To accomplish this, site-directed mutagenesis will be used in combination with solution Nuclear Magnetic Resonance (NMR) relaxation techniques. Two protein systems are chosen for the proposed study. The first system, calmodulin, is a signaling protein that shows allosteric coupling between its calcium- and target-binding sites. Mutations in the vicinity of either binding site, known to affect calcium- or target-binding properties of calmodulin, will be introduced. The second system is drug-resistant mutants of HIV-1 protease, for which mutations distant from the active site were shown to be responsible for the impaired binding affinity towards protease inhibitors. Information about the backbone and sidechain dynamics of the mutant protein complexes with appropriate ligands will be obtained using NMR relaxation techniques. Dynamically perturbed residues will be identified by comparison with the wild-type protein complexes. The findings of this proposal will help to uncover the mechansim of allosteric coupling in monomeric proteins and the role of protein flexibility in the development of drug resistance.
| Igumenova, Tatyana I; Lee, Andrew L; Wand, A Joshua (2005) Backbone and side chain dynamics of mutant calmodulin-peptide complexes. Biochemistry 44:12627-39 |
