title: Weak interactions between folate and osmolytes in solution.
abstract: Previous osmotic stress studies on the role of solvent in two structurally unrelated dihydrofolate reductases (DHFRs) found weaker binding of dihydrofolate (DHF) to either enzyme in the presence of osmolytes. To explain these unusual results, weak interactions between DHF and osmolytes were proposed, with a competition between osmolyte and DHFR for DHF. High osmolyte concentrations will inhibit binding of the cognate pair. To evaluate this hypothesis, we devised a small molecule approach. Dimerization of folate, monitored by nuclear magnetic resonance, was weakened 2-3-fold upon addition of betaine or dimethyl sulfoxide (DMSO), supporting preferential interaction of either osmolyte with the monomer (as it possesses a larger surface area). Nuclear Overhauser effect (NOE) spectroscopy experiments found a positive NOE for the interaction of the C3'/C5' benzoyl ring protons with the C9 proton in buffer; however, a negative NOE was observed upon addition of betaine or DMSO. This change indicated a decreased tumbling rate, consistent with osmolyte interaction. Osmotic stress experiments also showed that betaine, DMSO, and sucrose preferentially interact with folate. Further, studies with the folate fragments, p-aminobenzoic acid and pterin 6-carboxylate, revealed interactions for both model compounds with betaine and sucrose. In contrast, DMSO was strongly excluded from the pterin ring but preferentially interacted with the p-aminobenzoyl moiety. These interactions are likely to be important in vivo because of the crowded conditions of the cell where weak contacts can more readily compete with specific binding interactions.
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, United States.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Duff
authors.forename: Jordan
authors.initials: J
authors.lastname: Grubbs
authors.forename: Engin
authors.initials: E
authors.lastname: Serpersu
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Howell
mesh_terms: D010129:4-Aminobenzoic Acid / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D019281:Dimerization; D004121:Dimethyl Sulfoxide / Q000737:chemistry / Q000378:metabolism; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009994:Osmolar Concentration; D011621:Pteridines; D012996:Solutions; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism
chemical_list: D011621:Pteridines; D012996:Solutions; C405202:pterine-6-carboxylic acid; C010920:dihydrofolate; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D010129:4-Aminobenzoic Acid; D004121:Dimethyl Sulfoxide