Influence of stabilizers cosolutes on catalase conformation

Belluzo, Soledad; Boeris, Valeria; Farruggia, Beatriz; Picó, Guillermo

doi:10.1016/j.ijbiomac.2011.08.012

Cited by 16 publications

(10 citation statements)

References 16 publications

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“…While osmolytes that are excluded from the protein surface stabilize proteins (e.g., trimethylamine oxide with chemically modified RNase T 1 , FolM with betaine), denaturants that interact with the protein (for example, urea) destabilize proteins. − DMSO, , as well as other osmolytes, has also been found to destabilize proteins. Lin and Timasheff propose it is the difference between association of the cosolvent with the native and denatured state that affects the stability of a protein compared to water .…”

Section: Discussionmentioning

confidence: 99%

Investigation of Osmolyte Effects on FolM: Comparison with Other Dihydrofolate Reductases

et al. 2014

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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.

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Section: Discussionmentioning

confidence: 99%

Investigation of Osmolyte Effects on FolM: Comparison with Other Dihydrofolate Reductases

et al. 2014

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“…These results indicate that osmolyte-protein interactions can also complicate the analysis of ligand binding. If there are variable slopes in plots of lnK a vs. osmolality, the effect of osmolytes on protein structure and stability can be examined by DSC to examine the possibility of osmolyte-protein interaction(s) [65–71]. …”

Section: 1 Osmotic Stress Experiments Probe the Role Of Watermentioning

confidence: 99%

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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“…Enzymes respond differently to crowding agents; the activity of some decreases and of others increases, and the extents differ as well. For example, while macro- molecular crowding has minimal effects on the kinetics and function of yeast hexokinase in physiological solutions [16], it can significantly increase the thermal stability of catalase; and the overall structure becomes more rigid [17]. Crowding effects are also concentration-dependent.…”

Section: Introductionmentioning

confidence: 99%

Structured Crowding and Its Effects on Enzyme Catalysis

Ma¹,

Nussinov²

2013

Topics in Current Chemistry

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Macromolecular crowding decreases the diffusion rate, shifts the equilibrium of protein-protein and protein-substrate interactions, and changes protein conformational dynamics. Collectively, these effects contribute to enzyme catalysis. Here we describe how crowding may bias the conformational change and dynamics of enzyme populations and in this way affect catalysis. Crowding effects have been studied using artificial crowding agents and in vivo-like environments. These studies revealed a correlation between protein dynamics and function in the crowded environment. We suggest that crowded environments be classified into uniform crowding and structured crowding. Uniform crowding represents random crowding conditions created by synthetic particles with a narrow size distribution. Structured crowding refers to the highly coordinated cellular environment, where proteins and other macromolecules are clustered and organized. In structured crowded environments the perturbation of protein thermal stability may be lower; however, it may still be able to modulate functions effectively and dynamically. Dynamic, allosteric enzymes could be more sensitive to cellular perturbations if their free energy landscape is flatter around the native state; on the other hand, if their free energy landscape is rougher, with high kinetic barriers separating deep minima, they could be more robust. Above all, cells are structured; and this holds both for the cytosol and for the membrane environment. The crowded environment is organized, which limits the search, and the crowders are not necessarily inert. More likely, they too transmit allosteric effects, and as such play important functional roles. Overall, structured cellular crowding may lead to higher enzyme efficiency and specificity.

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Influence of stabilizers cosolutes on catalase conformation

Cited by 16 publications

References 16 publications

Investigation of Osmolyte Effects on FolM: Comparison with Other Dihydrofolate Reductases

Investigation of Osmolyte Effects on FolM: Comparison with Other Dihydrofolate Reductases

Thermodynamics and solvent linkage of macromolecule–ligand interactions

Structured Crowding and Its Effects on Enzyme Catalysis

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