Modulating Enzyme Function via Dynamic Allostery within Biliverdin Reductase B.

The biliverdin reductase B (BLVRB) class of enzymes catalyze the NADPH-dependent reduction of multiple flavin substrates and are emerging as critical players in cellular redox regulation. However, the role of dynamics and allostery have not been addressed, prompting studies here that have revealed a position 15 Å away from the active site within human BLVRB (T164) that is inherently dynamic and can be mutated to control global micro-millisecond motions and function. By comparing the inherent dynamics through nuclear magnetic resonance (NMR) relaxation approaches of evolutionarily distinct BLVRB homologues and by applying our previously developed Relaxation And Single Site Multiple Mutations (RASSMM) approach that monitors both the functional and dynamic effects of multiple mutations to the single T164 site, we have discovered that the most dramatic mutagenic effects coincide with evolutionary changes and these modulate coenzyme binding. Thus, evolutionarily changing sites distal to the active site serve as dynamic "dials" to globally modulate motions and function. Despite the distal dynamic and functional coupling modulated by this site, micro-millisecond motions span an order of magnitude in their apparent kinetic rates of motions. Thus, global dynamics within BLVRB are a collection of partially coupled motions tied to catalytic function.

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19 F NMR.

R67 dihydrofolate reductase (R67 DHFR) is a plasmid-encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry-related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N-termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N-terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4- and 5-fluoroindole labeled protein and severe line broadening for 6- and 7-fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the 19 F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities.

The Structural Basis for Nonsteroidal Anti-Inflammatory Drug Inhibition of Human Dihydrofolate Reductase.

Although nonsteroidal anti-inflammatory drugs (NSAIDs) target primarily cyclooxygenase enzymes, a subset of NSAIDs containing carboxylate groups also has been reported to competitively inhibit dihydrofolate reductase (DHFR). In this study, we have characterized NSAID interactions with human DHFR based on kinetic, NMR, and X-ray crystallographic methods. The NSAIDs target a region of the folate binding site that interacts with the p-aminobenzoyl-l-glutamate (pABG) moiety of folate and inhibit cooperatively with ligands that target the adjacent pteridine-recognition subsite. NSAIDs containing benzoate or salicylate groups were identified as having the highest potency. Among those tested, diflunisal, a salicylate derivative not previously identified to have anti-folate activity, was found to have a Ki of 34 µM, well below peak plasma diflunisal levels reached at typical dosage levels. The potential of these drugs to interfere with the inflammatory process by multiple pathways introduces the possibility of further optimization to design dual-targeted analogs.

Effects of Osmolytes on Ligand Binding to Dihydropteroate Synthase from Bacillus anthracis.

Osmolyte interactions with ligands can affect their affinity for proteins and are dependent upon the cosolute and the functional groups of the ligand. Here, we explored ligand binding to Bacillus anthracis dihydropteroate synthase (BaDHPS) under osmotic stress conditions. Osmolyte effects were specific to the cosolute and ligand, suggesting interaction of the osmolytes with the free ligands in solution. The association rates of pterin pyrophosphate were mostly unaffected by the osmolytes, except for a 2-fold decrease in the presence of 1 M trehalose, while the dissociation rates decreased in most osmolyte solutions. The viscosity and dielectric constant of the solution did not correlate with the effects of the osmolytes. Experimental results were compared with predicted preferential interaction coefficients (Δµ23/RT) between the osmolytes and ligands. The Δµ23/RT were able to predict the experimental data for most of the osmolytes. Trehalose and proline effects did not correlate with the predicted values, indicating that these two osmolytes may affect binding in more complex ways than simple preferential interactions. Additionally, osmolytes weakly interacted with the sulfa drug sulfathiazole, which altered its affinity for BaDHPS, suggesting that these types of weak interactions can also impact drug binding. As osmolytes affect ligands binding to two different folate cycle enzymes (DHFRs and DHPS), we predicted how ligand binding to other folate cycle enzymes will be altered by the presence of osmolytes.

Catalytic activity and stabilization of phenyl-modified glucose oxidase at high hydrostatic pressure.

Glucose oxidase (GOx) was modified by attaching phenyl groups to either carboxyl or amino side chains on the enzyme. High hydrostatic pressure (HHP) stabilized the aniline-, and benzoate-modified GOx at 69.1-80 °C compared to atmospheric pressure. At 240 MPa and 80.0 °C, the first order rate constant of inactivation kinact. of aniline-modified GOx was 20 × 10-2 min-1, or 3.7 times smaller than for the native GOx, while the kinact for benzoate-modified GOx was 26 × 10-2 min-1, or 2.8 times smaller than for the native GOx at the same temperature. Furthermore, at 240 MPa and 80.0 °C, the kinact of the aniline-modified GOx was 69 times smaller than the kinact of native GOx (1530 × 10-2 min-1) at 0.1 MPa and 80.0 °C. Similar results were obtained for benzoate-modified GOx. At each temperature in this study (25-69.1 °C), the catalytic activity of the native, aniline-, or benzoate-modified GOx increased with HHP, and reached a maximum at around 180 MPa. At 180 MPa and 69.1 °C, aniline-modified GOx produced the fastest catalytic rate, followed by benzoate-modified GOx, and then native GOx. An increase in temperature increased the activation volume of the reaction. Similarly, the activation energy increased with pressure. The combination of HHP and hydrophobic modification made GOx more thermostable and increased the effect of temperature in enzyme activity.

Structure, dynamics and function of the evolutionarily changing biliverdin reductase B family.

Biliverdin reductase B (BLVRB) family members are general flavin reductases critical in maintaining cellular redox with recent findings revealing that BLVRB alone can dictate cellular fate. However, as opposed to most enzymes, the BLVRB family remains enigmatic with an evolutionarily changing active site and unknown structural and functional consequences. Here, we applied a multi-faceted approach that combines X-ray crystallography, NMR and kinetics methods to elucidate the structural and functional basis of the evolutionarily changing BLVRB active site. Using a panel of three BLVRB isoforms (human, lemur and hyrax) and multiple human BLVRB mutants, our studies reveal a novel evolutionary mechanism where coenzyme 'clamps' formed by arginine side chains at two co-evolving positions within the active site serve to slow coenzyme release (Positions 14 and 78). We find that coenzyme release is further slowed by the weaker binding substrate, resulting in relatively slow turnover numbers. However, different BLVRB active sites imposed by either evolution or mutagenesis exhibit a surprising inverse relationship between coenzyme release and substrate turnover that is independent of the faster chemical step of hydride transfer also measured here. Collectively, our studies have elucidated the role of the evolutionarily changing BLVRB active site that serves to modulate coenzyme release and has revealed that coenzyme release is coupled to substrate turnover.

Crowders Steal Dihydrofolate Reductase Ligands through Quinary Interactions.

Dihydrofolate reductase (DHFR) reduces dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. Due to its role in one carbon metabolism, chromosomal DHFR is the target of the antibacterial drug, trimethoprim. Resistance to trimethoprim has resulted in a type II DHFR that is not structurally related to the chromosomal enzyme target. Because of its metabolic significance, understanding DHFR kinetics and ligand binding behavior in more cell-like conditions, where the total macromolecule concentration can be as great as 300 mg/mL, is important. The progress-curve kinetics and ligand binding properties of the drug target (chromosomal E. coli DHFR) and the drug resistant (R67 DHFR) enzymes were studied in the presence of macromolecular cosolutes. There were varied effects on NADPH oxidation and binding to the two DHFRs, with some cosolutes increasing affinity and others weakening binding. However, DHF binding and reduction in both DHFRs decreased in the presence of all cosolutes. The decreased binding of ligands is mostly attributed to weak associations with the macromolecules, as opposed to crowder effects on the DHFRs. Computer simulations found weak, transient interactions for both ligands with several proteins. The net charge of protein cosolutes correlated with effects on NADP+ binding, with near neutral and positively charged proteins having more detrimental effects on binding. For DHF binding, effects correlated more with the size of binding pockets on the protein crowders. These nonspecific interactions between DHFR ligands and proteins predict that the in vivo efficiency of DHFRs may be much lower than expected from their in vitro rates.

In Vivo Titration of Folate Pathway Enzymes.

How enzymes behave in cells is likely different from how they behave in the test tube. Previous in vitro studies find that osmolytes interact weakly with folate. Removal of the osmolyte from the solvation shell of folate is more difficult than removal of water, which weakens binding of folate to its enzyme partners. To examine if this phenomenon occurs in vivo, osmotic stress titrations were performed with Escherichia coli Two strategies were employed: resistance to an antibacterial drug and complementation of a knockout strain by the appropriate gene cloned into a plasmid that allows tight control of expression levels as well as labeling by a degradation tag. The abilities of the knockout and complemented strains to grow under osmotic stress were compared. Typically, the knockout strain could grow to high osmolalities on supplemented medium, while the complemented strain stopped growing at lower osmolalities on minimal medium. This pattern was observed for an R67 dihydrofolate reductase clone rescuing a ΔfolA strain, for a methylenetetrahydrofolate reductase clone rescuing a ΔmetF strain, and for a serine hydroxymethyltransferase clone rescuing a ΔglyA strain. Additionally, an R67 dihydrofolate reductase clone allowed E. coli DH5α to grow in the presence of trimethoprim until an osmolality of ∼0.81 is reached, while cells in a control titration lacking antibiotic could grow to 1.90 osmol.IMPORTANCEE. coli can survive in drought and flooding conditions and can tolerate large changes in osmolality. However, the cell processes that limit bacterial growth under high osmotic stress conditions are not known. In this study, the dose of four different enzymes in E. coli was decreased by using deletion strains complemented by the gene carried in a tunable plasmid. Under conditions of limiting enzyme concentration (lower than that achieved by chromosomal gene expression), cell growth can be blocked by osmotic stress conditions that are normally tolerated. These observations indicate that E. coli has evolved to deal with variations in its osmotic environment and that normal protein levels are sufficient to buffer the cell from environmental changes. Additional factors involved in the osmotic pressure response may include altered protein concentration/activity levels, weak solute interactions with ligands which can make it more difficult for proteins to bind their substrates/inhibitors/cofactors in vivo, and/or viscosity effects.

Modulating Enzyme Activity by Altering Protein Dynamics with Solvent.

Optimal enzyme activity depends on a number of factors, including structure and dynamics. The role of enzyme structure is well recognized; however, the linkage between protein dynamics and enzyme activity has given rise to a contentious debate. We have developed an approach that uses an aqueous mixture of organic solvent to control the functionally relevant enzyme dynamics (without changing the structure), which in turn modulates the enzyme activity. Using this approach, we predicted that the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) from Escherichia coli in aqueous mixtures of isopropanol (IPA) with water will decrease by ∼3 fold at 20% (v/v) IPA concentration. Stopped-flow kinetic measurements find that the pH-independent khydride rate decreases by 2.2 fold. X-ray crystallographic enzyme structures show no noticeable differences, while computational studies indicate that the transition state and electrostatic effects were identical for water and mixed solvent conditions; quasi-elastic neutron scattering studies show that the dynamical enzyme motions are suppressed. Our approach provides a unique avenue to modulating enzyme activity through changes in enzyme dynamics. Further it provides vital insights that show the altered motions of DHFR cause significant changes in the enzyme's ability to access its functionally relevant conformational substates, explaining the decreased khydride rate. This approach has important implications for obtaining fundamental insights into the role of rate-limiting dynamics in catalysis and as well as for enzyme engineering.

Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N-Termini.

R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of Escherichia coli DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE ( http://www.smallangles.net/sassie/SASSIE_HOME.html ), the apoenzyme displays a radius of gyration (Rg) of 21.46 ± 0.50 Å. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the Rg increases slightly to 22.78 ± 0.87 Å. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and remain disordered, suggesting they might function as entropic bristles. Pressure perturbation calorimetry also indicated that the volume of R67 DHFR increases upon addition of 10% betaine and decreased at 20% betaine because of the dehydration of the protein. Studies of the hydration of full-length R67 DHFR in the presence of the osmolytes betaine and dimethyl sulfoxide find around 1250 water molecules hydrating the protein. Similar studies with truncated R67 DHFR yield around 400 water molecules hydrating the protein in the presence of betaine. The difference of ∼900 waters indicates the N-termini are well-hydrated.

A Structural Basis for Biguanide Activity.

Metformin is the most commonly prescribed treatment for type II diabetes and related disorders; however, molecular insights into its mode(s) of action have been limited by an absence of structural data. Structural considerations along with a growing body of literature demonstrating its effects on one-carbon metabolism suggest the possibility of folate mimicry and anti-folate activity. Motivated by the growing recognition that anti-diabetic biguanides may act directly upon the gut microbiome, we have determined structures of the complexes formed between the anti-diabetic biguanides (phenformin, buformin, and metformin) and Escherichia coli dihydrofolate reductase (ecDHFR) based on nuclear magnetic resonance, crystallographic, and molecular modeling studies. Interligand Overhauser effects indicate that metformin can form ternary complexes with p-aminobenzoyl-l-glutamate (pABG) as well as other ligands that occupy the region of the folate-binding site that interacts with pABG; however, DHFR inhibition is not cooperative. The biguanides competitively inhibit the activity of ecDHFR, with the phenformin inhibition constant being 100-fold lower than that of metformin. This inhibition may be significant at concentrations present in the gut of treated individuals, and inhibition of DHFR in intestinal mucosal cells may also occur if accumulation levels are sufficient. Perturbation of folate homeostasis can alter the pyridine nucleotide redox ratios that are important regulators of cellular metabolism.

Effects of high hydrostatic pressure or hydrophobic modification on thermal stability of xanthine oxidase.

The effect of high hydrostatic pressure (HHP) on the kinetics of thermal inactivation of xanthine oxidase (XOx) from bovine milk was studied. Inactivation of XOx followed pseudo-first-order kinetics at 0.1-300MPa and 55.0-70.0°C. High pressure up to at least 300MPa stabilized XOx at all the studied temperatures. The highest stabilization effect of HHP on XOx was at 200-300MPa at 55.0 and 58.6°C, and at 250-300MPa at 62.3-70.0°C. The stability of XOx increased 9.5 times at 300MPa and 70.0°C compared to atmospheric pressure at the same temperature. The activation energy of inactivation of XOx decreased with pressure and was 1.9 times less at 300MPa (97.0±8.2kJmol-1) than at 0.1MPa (181.7±12.1kJmol-1). High pressure decreased the dependence of the rate constant of inactivation to temperature effects compared to atmospheric pressure. The stabilizing effect of HHP on XOx was highest at 70.0°C where the activation volume of inactivation of XOx was 28.9±2.9cm3mol-1. A second approach to try to increase XOx stability involved hydrophobic modification using aniline or benzoate. However, the thermal stability of XOx remained unaffected after 8-14 modifications of carboxyl side groups per XOx monomer with aniline, or 12-17 modifications of amino side groups per XOx monomer with benzoate.

Glucose oxidase stabilization against thermal inactivation using high hydrostatic pressure and hydrophobic modification.

High hydrostatic pressure (HHP) stabilized glucose oxidase (GOx) against thermal inactivation. The apparent first-order kinetics of inactivation of GOx were investigated at 0.1-300 MPa and 58.8-80.0°C. At 240 MPa and 74.5°C, GOx inactivated at a rate 50 times slower than at atmospheric pressure at the same temperature. The apparent activation energy of inactivation at 300 MPa was 281.0 ± 17.4 kJ mol-1 or 1.3-fold smaller than for the inactivation at atmospheric pressure (378.1 ± 25.6 kJ mol-1 ). The stabilizing effect of HHP was greatest at 74.5°C, where the activation volume of 57.0 ± 12.0 cm3 mol-1 was highest compared to all other studied temperatures. Positive apparent activation volumes for all the treatment temperatures confirmed that HHP favors GOx stabilization. A second approach to increase GOx stability involved crosslinking with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and either aniline or benzoate. The modified enzyme remained fully active with only slight increases in KM (1.3-1.9-fold increases for aniline and benzoate modification, respectively). The thermal stability of GOx increased by 8°C with aniline modification, while it decreased by 0.9°C upon modification with benzoate. Biotechnol. Bioeng. 2017;114: 516-525. © 2016 Wiley Periodicals, Inc.

Aspects of Weak Interactions between Folate and Glycine Betaine.

Folate, or vitamin B9, is an important compound in one-carbon metabolism. Previous studies have found weaker binding of dihydrofolate to dihydrofolate reductase in the presence of osmolytes. In other words, osmolytes are more difficult to remove from the dihydrofolate solvation shell than water; this shifts the equilibrium toward the free ligand and protein species. This study uses vapor-pressure osmometry to explore the interaction of folate with the model osmolyte, glycine betaine. This method yields a preferential interaction potential (µ23/RT value). This value is concentration-dependent as folate dimerizes. The µ23/RT value also tracks the deprotonation of folate's N3-O4 keto-enol group, yielding a pKa of 8.1. To determine which folate atoms interact most strongly with betaine, the interaction of heterocyclic aromatic compounds (as well as other small molecules) with betaine was monitored. Using an accessible surface area approach coupled with osmometry measurements, deconvolution of the µ23/RT values into α values for atom types was achieved. This allows prediction of µ23/RT values for larger molecules such as folate. Molecular dynamics simulations of folate show a variety of structures from extended to L-shaped. These conformers possess µ23/RT values from -0.18 to 0.09 m-1, where a negative value indicates a preference for solvation by betaine and a positive value indicates a preference for water. This range of values is consistent with values observed in osmometry and solubility experiments. As the average predicted folate µ23/RT value is near zero, this indicates folate interacts almost equally well with betaine and water. Specifically, the glutamate tail prefers to interact with water, while the aromatic rings prefer betaine. In general, the more protonated species in our small molecule survey interact better with betaine as they provide a source of hydrogens (betaine is not a hydrogen bond donor). Upon deprotonation of the small molecule, the preference swings toward water interaction because of its hydrogen bond donating capacities.

Highly Dynamic Anion-Quadrupole Networks in Proteins.

The dynamics of anion-quadrupole (or anion-π) interactions formed between negatively charged (Asp/Glu) and aromatic (Phe) side chains are for the first time computationally characterized in RmlC (Protein Data Bank entry 1EP0 ), a homodimeric epimerase. Empirical force field-based molecular dynamics simulations predict anion-quadrupole pairs and triplets (anion-anion-π and anion-π-π) are formed by the protein during the simulated trajectory, which suggests that the anion-quadrupole interactions may provide a significant contribution to the overall stability of the protein, with an average of -1.6 kcal/mol per pair. Some anion-π interactions are predicted to form during the trajectory, extending the number of anion-quadrupole interactions beyond those predicted from crystal structure analysis. At the same time, some anion-π pairs observed in the crystal structure exhibit marginal stability. Overall, most anion-π interactions alternate between an "on" state, with significantly stabilizing energies, and an "off" state, with marginal or null stabilizing energies. The way proteins possibly compensate for transient loss of anion-quadrupole interactions is characterized in the RmlC aspartate 84-phenylalanine 112 anion-quadrupole pair observed in the crystal structure. A double-mutant cycle analysis of the thermal stability suggests a possible loss of anion-π interactions compensated by variations of hydration of the residues and formation of compensating electrostatic interactions. These results suggest that near-planar anion-quadrupole pairs can exist, sometimes transiently, which may play a role in maintaining the structural stability and function of the protein, in an otherwise very dynamic interplay of a nonbonded interaction network as well as solvent effects.

Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase.

Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ∼35% were constructed. Only minor effects on k(cat) were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The K(i) for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis.

Thermodynamics and solvent linkage of macromolecule-ligand interactions.

Binding involves two steps, desolvation and association. While water is ubiquitous and occurs at high concentration, it is typically ignored. In vitro experiments typically use infinite dilution conditions, while in vivo, the concentration of water is decreased due to the presence of high concentrations of molecules in the cellular milieu. This review discusses isothermal titration calorimetry approaches that address the role of water in binding. For example, use of D2O allows the contribution of solvent reorganization to the enthalpy component to be assessed. Further, the addition of osmolytes will decrease the water activity of a solution and allow effects on Ka to be determined. In most cases, binding becomes tighter in the presence of osmolytes as the desolvation penalty associated with binding is minimized. In other cases, the osmolytes prefer to interact with the ligand or protein, and if their removal is more difficult than shedding water, then binding can be weakened. These complicating layers can be discerned by different slopes in ln(Ka) vs osmolality plots and by differential scanning calorimetry in the presence of the osmolyte.

Investigation of osmolyte effects on FolM: comparison with other dihydrofolate reductases.

A weak association between osmolytes and dihydrofolate (DHF) decreases the affinity of the substrate for the Escherichia coli chromosomal and R67 plasmid dihydrofolate reductase (DHFR) enzymes. To test whether the osmolyte-DHF association also interferes with binding of DHF to FolM, an E. coli enzyme that possesses weak DHFR activity, ligand binding was monitored in the presence of osmolytes. The affinity of FolM for DHF, measured by kcat/Km(DHF), was decreased by the addition of an osmolyte. Additionally, binding of the antifolate drug, methotrexate, to FolM was weakened by the addition of an osmolyte. The changes in ligand binding with water activity were unique for each osmolyte, indicating preferential interaction between the osmolyte and folate and its derivatives; however, additional evidence provided support for further interactions between FolM and osmolytes. Binding of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) cofactor to FolM was monitored by isothermal titration calorimetry as a control for protein-osmolyte association. In the presence of betaine (proposed to be the osmolyte most excluded from protein surfaces), the NADPH Kd decreased, consistent with dehydration effects. However, other osmolytes did not tighten binding to the cofactor. Rather, dimethyl sulfoxide (DMSO) had no effect on the NADPH Kd, while ethylene glycol and polyethylene glycol 400 weakened cofactor binding. Differential scanning calorimetry of FolM in the presence of osmolytes showed that both DMSO and ethylene glycol decreased the stability of FolM, while betaine increased the stability of the protein. These results suggest that some osmolytes can destabilize FolM by preferentially interacting with the protein. Further, these weak attractions can impede ligand binding. These various contributions have to be considered when interpreting osmotic pressure results.

Further studies on the role of water in R67 dihydrofolate reductase.

Previous osmotic pressure studies of two nonhomologous dihydrofolate reductase (DHFR) enzymes found tighter binding of the nicotinamide adenine dinucleotide phosphate cofactor upon addition of neutral osmolytes. This result is consistent with water release accompanying binding. In contrast, osmotic stress studies found weaker binding of the dihydrofolate (DHF) substrate for both type I and type II DHFRs in the presence of osmolytes; this observation can be explained if dihydrofolate interacts with osmolytes and shifts the equilibrium from the enzyme-bound state toward the unbound substrate. Nuclear magnetic resonance experiments support this hypothesis, finding that osmolytes interact with dihydrofolate. To consider binding without added osmolytes, a high-pressure approach was used. In this study, the type II enzyme, R67 DHFR, was subjected to high hydrostatic pressure (HHP). Both enzyme activity and fluorescence measurements find the protein tolerates pressures up to 200 MPa. Binding of the cofactor to R67 DHFR weakens with increasing pressure, and a positive association volume of 11.4 ± 0.5 cm(3)/mol was measured. Additionally, an activation volume of 3.3 ± 0.5 cm(3)/mol describing k(cat)/K(m(DHF)) was determined from progress curve analysis. Results from these HHP experiments suggest water release accompanies binding of both the cofactor and DHF to R67 DHFR. In an additional set of experiments, isothermal titration calorimetry studies in H2O and D2O find that water reorganization dominates the enthalpy associated with binding of DHF to R67 DHFR·NADP(+), while no obvious effects occur for cofactor binding. The combined results indicate that water plays an active role in ligand binding to R67 DHFR.

Weak interactions between folate and osmolytes in solution.

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.