Towards Understanding Osmolyte Effects on Folate(s) and Dihydrofolate Reductase Proteins

Osmolytes are small molecules that alter water activity and probe role of water in biological processes. Osmotic stress approach explored the role of water in ligand binding to dihydrofolate reductase (DHFR). DHFR catalyzes NADPH dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), which is essential for the synthesis of DNA, amino acids and other metabolic intermediates. R67 DHFR is a plasmid-encoded DHFR that confers resistance against trimethoprim, which is a potent inhibitor of E.coli chromosomal DHFR.

Osmolytes addition decreases the affinity of the substrate towards both the DHFRs. Weak preferential interactions between the osmolytes and DHF impede substrate binding to the enzyme. Similar results were obtained for DHF binding to FolM, an E. coli enzyme which possesses weak DHFR activity. Binding of the cofactor to FolM was found to be tighter in presence of betaine but other osmolytes showed variable effects indicating interactions between FolM and osmolytes. Osmolytes (DMSO and ethylene glycol) showed decreased the stability of FolM further suggesting preferential interactions of osmolytes with the protein. Thus, ligand binding to FolM was hindered by interactions between osmolytes and the enzyme as well as the substrate.

Interaction potential (μ₂₃/RT value) of folate with betaine was quantified using a vapor pressure osmometry method. Folate interaction with betaine showed concentration dependence as folate dimerizes. A pH dependence owing to the deprotonation of folate’s N3-O4 keto-enol group was also seen. The interaction of other heterocyclic aromatic compounds with betaine was monitored and deconvoluted into atomistic interaction potentials using an accessible surface area approach. Betaine preferentially interact witharomatic surfaces, cationic and amide nitrogens whereas it is excluded from carboxylate oxygens and aromatic nitrogens. As folate contains a combination of surface types, the μ₂₃/RT value is predicted to be near zero, indicating folate interacts almost equally well with betaine and water.

Further, osmolyte effect on proteins was explored using SANS studies on R67 DHFR. The hydration studies yielded around 1200 water molecules excluding osmolytes from R67 DHFR surface. SANS also characterized the conformations sampled by the disordered tails of R67 DHFR under different conditions tested.