Do soft anions promote protein denaturation through binding interactions? A case study using ribonuclease A

Francisco, Olga; Clark, Courtney; Glor, Hayden M.; Khajehpour, Mazdak

doi:10.1039/c8ra10303h

Cited by 12 publications

(33 citation statements)

References 81 publications

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“…Accordingly, to investigate whether the AmpC mutations affect enzyme stability, the melting temperatures (Tm) of the WT and mutant AmpC enzymes were measured. The Tm determined for WT AmpC (i.e., PDC-1 from P. aeruginosa PAO1) (55.33˚C) is comparable to those of other WT AmpC analogues, including E. coli AmpC (54.6˚C) (Beadle et al 1999) (Francisco et al 2019), where ∆Hunfolding is the unfolding enthalpy, which is positive during thermal denaturation (Pica and Graziamo 2016), and Tm is the melting temperature of the WT AmpC enzyme. That is to say, these mutations decrease the stability of AmpC which in turn increases its flexibility and dynamics at room temperature.…”

Section: Relationship Between Ampc Stability Flexibility and Activitymentioning

confidence: 67%

Adding Insult to Injury: Mechanistic Basis for How AmpC Mutations Allow Pseudomonas aeruginosa To Accelerate Cephalosporin Hydrolysis and Evade Avibactam

Slater

Winogrodzki

Fraile-Ribot

et al. 2020

Antimicrob Agents Chemother

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Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide and notorious for its broad-spectrum resistance to antibiotics. A key mechanism that provides extensive resistance to β-lactam antibiotics is the inducible expression of AmpC β-lactamase. Recently, a number of clinical isolates expressing mutated forms of AmpC have been found to be clinically resistant to the antipseudomonal β-lactam/β-lactamase inhibitor (BLI) combinations ceftolozane/tazobactam and ceftazidime/avibactam. Here, we compare the enzymatic activity of wild-type (WT) AmpC from PAO1 to four of these reported AmpC mutants, E247K, G183D, T96I, and ΔG229–E247, to gain detailed insights into how these mutations circumvent these clinically vital antibiotic/inhibitor combinations. We found that these mutations exert a twofold effect on the catalytic cycle of AmpC. First, they reduce the stability of the enzyme, thereby increasing its flexibility. This appears to increase the rate of deacylation of the enzyme-bound β-lactam, resulting in greater catalytic efficiencies towards ceftolozane and ceftazidime. Second, these mutations reduce the affinity of avibactam for AmpC by increasing the apparent activation barrier of the enzyme acylation step. This does not influence the catalytic turnover of ceftolozane and ceftazidime significantly, as deacylation is the rate-limiting step for the breakdown of these antibiotic substrates. It is remarkable that these mutations enhance the catalytic efficiency of AmpC towards ceftolozane and ceftazidime while simultaneously reducing susceptibility to inhibition by avibactam. Knowledge gained from the molecular analysis of these and other AmpC resistance mutants we believe will help aid the design of β-lactams and BLIs with reduced susceptibility to mutational resistance.

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Section: Relationship Between Ampc Stability Flexibility and Activitymentioning

confidence: 67%

Adding Insult to Injury: Mechanistic Basis for How AmpC Mutations Allow Pseudomonas aeruginosa To Accelerate Cephalosporin Hydrolysis and Evade Avibactam

Slater

Winogrodzki

Fraile-Ribot

et al. 2020

Antimicrob Agents Chemother

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“…The aqueous solubility of hydrophobic molecules is particularly sensitive to salt addition and this property has found use in diverse chemical processes such as aqueous phase organic chemistry, salting‐out extraction, hydrophobic interaction chromatography and the synthesis of colloids and membrane vesicles . The presence of salt also affects the thermodynamic properties of biological macromolecules and it has been demonstrated that ion‐specific changes in the solubility of hydrophobic protein moieties is a major contributor to salt induced perturbations to the overall protein folding free energy . This being said, we still lack a complete mechanistic picture of how the presence of ions modulates the solubility of hydrophobic molecules.…”

Section: Introductionmentioning

confidence: 99%

Salt Effects on Hydrophobic Solvation: Is the Observed Salt Specificity the Result of Excluded Volume Effects or Water Mediated Ion‐Hydrophobe Association?

2020

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The solubility of hydrophobic molecules in water is sensitive to salt addition in an ion‐specific manner. Such “salting‐out” and “salting‐in” properties have been shown to be a major contributor to the measured ion‐specific Hofmeister effects that are observed in many biophysical phenomena. Various theoretical models have suggested a number of disparate mechanisms for salting‐out (salting‐in) of hydrophobic moieties, the most popular of which include preferential interaction, water‐mediated association, and electrostriction models. However, a complete molecular level description of this ion‐specificity is not yet available. This work investigates the ion‐specific nature of hydrophobic solvation by studying how sodium and chloride salts affect the thermodynamics of 1,2‐hexanediol micellization. The results of this study are analyzed in terms of scaled‐particle theory and we show that salt addition can affect hydrophobic solvation in two modalities: salt addition changes the cavitation free energy; salt addition also influences the solvent‐solute interaction energy by changing the hydration of the hydrophobic solute. These two effects are salt specific in nature and we suggest that for small hydrophobic solutes these effects are the main cause of salt‐specific Hofmeister effects on their solubility.

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“…ref. 18 ). A fact overlooked in later expositions of this theory is the linear dependence of the specific heat Δ c p on salt concentration.…”

Section: Introductionmentioning

confidence: 99%

Denaturation of proteins: electrostatic effects vs. hydration

Ballauff

2022

RSC Adv.

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Do soft anions promote protein denaturation through binding interactions? A case study using ribonuclease A

Abstract: Soft anions promote protein folding through binding backbone CH and CH2 groups.

Cited by 12 publications

References 81 publications

Adding Insult to Injury: Mechanistic Basis for How AmpC Mutations Allow Pseudomonas aeruginosa To Accelerate Cephalosporin Hydrolysis and Evade Avibactam

Adding Insult to Injury: Mechanistic Basis for How AmpC Mutations Allow Pseudomonas aeruginosa To Accelerate Cephalosporin Hydrolysis and Evade Avibactam

Salt Effects on Hydrophobic Solvation: Is the Observed Salt Specificity the Result of Excluded Volume Effects or Water Mediated Ion‐Hydrophobe Association?

Denaturation of proteins: electrostatic effects vs. hydration

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