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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.

Chromosomal dihydrofolate reductase from Escherichia coli catalyzes the reduction of dihydrofolate to tetrahydrofolate using NADPH as a cofactor. The thermodynamics of ligand binding were examined using an isothermal titration calorimetry approach. Using buffers with different heats of ionization, zero to a small, fractional proton release was observed for dihydrofolate binding, while a proton was released upon NADP(+) binding. The role of water in binding was additionally monitored using a number of different osmolytes. Binding of NADP(+) is accompanied by the net release of ∼5-24 water molecules, with a dependence on the identity of the osmolyte. In contrast, binding of dihydrofolate is weakened in the presence of osmolytes, consistent with "water uptake". Different effects are observed depending on the identity of the osmolyte. The net uptake of water upon dihydrofolate binding was previously observed in the nonhomologous R67-encoded dihydrofolate reductase (dfrB or type II enzyme) [Chopra, S., et al. (2008) J. Biol. Chem. 283, 4690-4698]. As R67 dihydrofolate reductase possesses a nonhomologous sequence and forms a tetrameric structure with a single active site pore, the observation of weaker DHF binding in the presence of osmolytes in both enzymes implicates cosolvent effects on free dihydrofolate. Consistent with this analysis, stopped flow experiments find betaine mostly affects DHF binding via changes in k(on), while betaine mostly affects NADPH binding via changes in k(off). Finally, nonadditive enthalpy terms when binary and ternary cofactor binding events are compared suggest the presence of long-lived conformational transitions that are not included in a simple thermodynamic cycle.

R67 dihydrofolate reductase (DHFR) is a plasmid-encoded, type II enzyme. Four monomers (78 amino acids long) assemble into a homotetramer possessing 222 symmetry. In previous studies, a tandem array of four R67 DHFR gene copies was fused in frame to generate a functional monomer named Quad1. This protein possessed the essential tertiary structure of the R67 "parent". To facilitate mutagenesis reactions, restriction enzyme sites were introduced in the tandem gene array. S59A and H362L mutations were also added to minimize possible folding topologies; this protein product, named Quad3, possesses 10 substitutions and is functional. Since R67 DHFR possesses a stable scaffold, a large jump in sequence space was performed by the further addition of 45 amino acid substitutions. The mutational design utilized alternate sequences from other type II DHFRs. In addition, most of the mutations were positioned on the surface of the protein as well as in the disordered N-terminal sequence, which serves as the linker between the fused domains. The resulting Quad4 protein is quite functional; however, it is less stable than Quad1, suffering a DeltaDeltaG loss of 5 kcal/mol at pH 5. One unexpected result was formation of Quad4 dimers and higher order oligomers at pH 8. R67 DHFR, and its derivative Quad proteins, possesses a robust scaffold, capable of withstanding introduction ofor=55 substitutions.

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.