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.

Biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm

“…Once metabolites are bound on the surface of a macromolecule, diffusion becomes two-dimensional and slows down considerably as illustrated in detail for ATP and valine (Figure 6C; Video 4). Trapping of metabolites on macromolecular surfaces reduces the effective concentration of freely-diffusing metabolites consistent with recent experiments that have inferred a large fraction of surface-interacting metabolites due to crowding (Duff et al, 2012). A recent analysis of absolute metabolite concentrations in E. coli has found that most concentrations were above the values of the Michaelis constant K m for enzymes binding those metabolites (Bennett et al, 2009).…”

Aspects of Weak Interactions between Folate and Glycine Betaine

“…76 We have also used nuclear magnetic resonance to observe interactions between folate and osmolytes (middle column, green equilibrium arrows). 15 Both sets of results are consistent with this model.…”

“…Homonuclear ( 1 H) nuclear Overhauser effect spectroscopy (NOESY) experiments found NOEs between the protons on the C7 atom of folate and protons on the C9 and C3′/C5′ atoms, and between the C9 proton and the C3′/C5′ protons (see Figure S1 for the structure and atom numbering of folate). 15 However, a change in sign for the NOE between the C9 and C3′/C5′ protons from positive (without betaine) to negative (with betaine) was observed. The change in the sign for the NOE suggested a slower rotational rate for the p- amino-benzoyl ring protons, indicative of an interaction between betaine and this ring.…”

“…The change in the sign for the NOE suggested a slower rotational rate for the p- amino-benzoyl ring protons, indicative of an interaction between betaine and this ring. 15 As folate has limited protons on its pterin ring, it is difficult to discern from our nuclear magnetic resonance (NMR) results if osmolytes interact with this moiety. Thus, we turned to alternate techniques.…”

“…15 To extend this study, we use vapor-pressure osmometry (VPO) to measure μ 23 / RT values, which are closely related to preferential interaction coefficients. The μ 23 / RT value measures the change in chemical potential of a small test compound with the change in molality of the osmolyte in solution.…”