Thermodynamics and Solvent Effects on Substrate and Cofactor Binding in <i>Escherichia coli</i> Chromosomal Dihydrofolate Reductase

Grubbs, Jordan; Rahmanian, Sharghi; DeLuca, Alexa; Padmashali, Chetan; Jackson, Michael J.; Duff, Michael R.; Howell, Elizabeth E.

doi:10.1021/bi2002373

Cited by 16 publications

(85 citation statements)

References 102 publications

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“…A similar set of results was observed. Binding of DHF to EcDHFR, as measured by ITC, was weaker in the presence of osmolytes compared to buffer alone, while NADPH binding was strengthened by addition of osmolytes [54]. Stopped-flow studies provided further insight into how osmolytes affected ligand binding.…”

Section: 1 Osmotic Stress Experiments Probe the Role Of Watermentioning

confidence: 99%

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Thermodynamics and solvent linkage of macromolecule–ligand interactions

Duff

Howell

2015

Methods

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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.

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Section: 1 Osmotic Stress Experiments Probe the Role Of Watermentioning

confidence: 99%

“…Typically, a structural technique, such as circular dichroism, is used to monitor any changes in the secondary structure of the biomacromolecule [54, 55, 62]. If there are changes in the biomacromolecule structure, then these structural changes will also affect the ITC results.…”

Section: 1 Conclusionmentioning

confidence: 99%

Thermodynamics and solvent linkage of macromolecule–ligand interactions

Duff

Howell

2015

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“…We solved for the free enzyme and ligand concentrations using thermodynamic equilibrium constants (Table S1) determined previously by isothermal titration calorimetry (ITC) for the interaction of DHFR with folate and DHFR with NADP + . 39 We were unable to find the necessary equilibrium constants for the binding of folate to DHFR·NADP + in the literature; therefore, we determined this value ourselves using ITC (Figure S2 and Table S1). …”

Section: Resultsmentioning

confidence: 99%

Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase

Reddish

Vaughn

et al. 2016

Biochemistry

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Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein–ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not.

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“…Previous studies have found that the in vitro addition of osmolytes to three different protein scaffolds that catalyze the dihydrofolate reductase (DHFR) reaction results in tighter binding of cofactor NADPH (8)(9)(10). Addition of osmolytes lowers the water activity of a solution; dehydration typically leads to tighter ligand binding, as less solvent needs to be removed to form a complex.…”

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confidence: 99%

“…Addition of osmolytes lowers the water activity of a solution; dehydration typically leads to tighter ligand binding, as less solvent needs to be removed to form a complex. In contrast, binding of DHF "breaks the rules," as weaker binding upon osmolyte addition is observed in these three different proteins (8)(9)(10). To model this behavior, we proposed that osmolytes weakly associate with DHF.…”

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In Vivo Titration of Folate Pathway Enzymes

Nambiar

Berhane

Shew

et al. 2018

Appl Environ Microbiol

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How enzymes behave in cells is likely different from how they behave in the test tube. Previous 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, osmotic stress titrations were performed with 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 Δ strain, for a methylenetetrahydrofolate reductase clone rescuing a Δ strain, and for a serine hydroxymethyltransferase clone rescuing a Δ strain. Additionally, an R67 dihydrofolate reductase clone allowed 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. 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 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 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 , and/or viscosity effects.

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Thermodynamics and Solvent Effects on Substrate and Cofactor Binding in Escherichia coli Chromosomal Dihydrofolate Reductase

Cited by 16 publications

References 102 publications

Thermodynamics and solvent linkage of macromolecule–ligand interactions

Thermodynamics and solvent linkage of macromolecule–ligand interactions

Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase

In Vivo Titration of Folate Pathway Enzymes

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