Exploration of Weak Interactions between Folate and Glycine-Betaine

Abstract

In vitro studies with two different dihydrofolate reductases (EcDHFR, E.coli chromosomal and R67 DHFR, plasmid encoded) have shown that weak interactions between osmolytes and the substrate, dihydrofolate, decreases its affinity towards these enzymes. The unique changes in binding affinity with water activity for each osmolyte indicate preferential interactions between osmolyte and folate and its derivatives. Characterization of these interactions is essential for better understanding of in vivo effects of folate and its various redox states with available functional groups inside the cell. Quantitation of weak interactions between folate and glycine-betaine using a vapor pressure osmometry method yields a preferential interaction coefficient, or μ23/RT value. This provides a scale for measuring the preference of folate for glycine-betaine relative to water. The predicted μ23/RT value for folate using an accessible surface area calculation indicates equal preference for water and glycine-betaine. Experimental measurements found a folate concentration dependence of the μ23/RT values, consistent with dimerization of folate. Studies with other model compounds suggest aromatic rings prefer to interact with glycine-betaine as compared to water. Our results also indicate neutral folate preferentially interacts with glycine-betaine whereas the anionic form excludes glycine-betaine.Can μ23/RT values be used to predict osmotic stress effects on ligand binding? In some cases, yes. However, the caveat is whether all the ligand atoms are used in binding. As glutamate excludes glycine-betaine, calculation of the μ23/RT value for polyglutamylated folates (pteroyltetra-γ-glutamate (PG4)) predicts an overall exclusion of glycine-betaine. This should translate into tighter binding of PG4 to DHFR. Our studies found glycine-betaine addition weakens binding of both folate and PG4 to R67 DHFR to similar extents. Similar effects were observed for folate binding to EcDHFR. These results indicate that the additional glutamates do not contribute to binding to DHFR.