title: Ligand binding to a high-energy partially unfolded protein.
abstract: The conformational energy landscape of a protein determines populations of all possible conformations of the protein and also determines the kinetics of the conversion between the conformations. Interaction with ligands influences the conformational energy landscapes of proteins and shifts populations of proteins in different conformational states. To investigate the effect of ligand binding on partial unfolding of a protein, we use Escherichia coli dihydrofolate reductase (DHFR) and its functional ligand NADP(+) as a model system. We previously identified a partially unfolded form of DHFR that is populated under native conditions. In this report, we determined the free energy for partial unfolding of DHFR at varying concentrations of NADP(+) and found that NADP(+) binds to the partially unfolded form as well as the native form. DHFR unfolds partially without releasing the ligand, though the binding affinity for NADP(+) is diminished upon partial unfolding. Based on known crystallographic structures of NADP(+) -bound DHFR and the model of the partially unfolded protein we previously determined, we propose that the adenosine-binding domain of DHFR remains folded in the partially unfolded form and interacts with the adenosine moiety of NADP(+) . Our result demonstrates that ligand binding may affect the conformational free energy of not only native forms but also high-energy non-native forms.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana, 47907.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Kasper
authors.forename: Chiwook
authors.initials: C
authors.lastname: Park
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D058767:Protein Unfolding; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D008024:Ligands; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase
keywords: DHFR; NADP+; ligand binding; partial unfolding; proteolysis