Low cost, microcontroller based heating device for multi-wavelength differential scanning fluorimetry

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters.

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“…3d ). The mutations did not introduce instability to the variants as judged from their “melting points” 57 (Supplementary Fig. 3e, f ).…”

Section: Resultsmentioning

confidence: 91%

“…A first derivative was calculated from this fit to obtain melting points as inflection points/maxima peaks 57 .…”

Section: Methodsmentioning

confidence: 99%

Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis

et al. 2022

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“…In the course of initiating the studies described here, we explicitly tested whether thermally-unfolded protein could be cooled, and then once again heated to yield the same thermal unfolding transition. In light of well-justified concerns about non-reversible unfolding and aggregation, a number of strategies have been proposed: these include using faster speeds for the unfolding process (to minimize the potential for aggregation) 65,66 and including in the reaction dyes that explicitly detect protein aggregation 67,68 .…”

Section: Discussionmentioning

confidence: 99%

Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities

Bai

Röder

Dickson

et al. 2019

Sci Rep

102

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Differential scanning fluorimetry (DSF), also known as ThermoFluor or Thermal Shift Assay, has become a commonly-used approach for detecting protein-ligand interactions, particularly in the context of fragment screening. Upon binding to a folded protein, most ligands stabilize the protein; thus, observing an increase in the temperature at which the protein unfolds as a function of ligand concentration can serve as evidence of a direct interaction. While experimental protocols for this assay are well-developed, it is not straightforward to extract binding constants from the resulting data. Because of this, DSF is often used to probe for an interaction, but not to quantify the corresponding binding constant (K d ). Here, we propose a new approach for analyzing DSF data. Using unfolding curves at varying ligand concentrations, our “isothermal” approach collects from these the fraction of protein that is folded at a single temperature (chosen to be temperature near the unfolding transition). This greatly simplifies the subsequent analysis, because it circumvents the complicating temperature dependence of the binding constant; the resulting constant-temperature system can then be described as a pair of coupled equilibria (protein folding/unfolding and ligand binding/unbinding). The temperature at which the binding constants are determined can also be tuned, by adding chemical denaturants that shift the protein unfolding temperature. We demonstrate the application of this isothermal analysis using experimental data for maltose binding protein binding to maltose, and for two carbonic anhydrase isoforms binding to each of four inhibitors. To facilitate adoption of this new approach, we provide a free and easy-to-use Python program that analyzes thermal unfolding data and implements the isothermal approach described herein ( https://sourceforge.net/projects/dsf-fitting ).

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“…The stability of the flavin site of NuoF was assessed using a “ThermoFAD” protocol 27 . The fluorescence of free flavin released from heat-denatured complex I and the variants were recorded with an RT-PCR machine (LightCycler 2.0; Roche, Germany) and a home-build machine 45 . The initial temperature of 30 °C was held for 5 min, and then increased by 1 °C every 20 s. To determine the “melting temperature” the first derivative of the original curve was calculated.…”

Section: Methodsmentioning

confidence: 99%

A mechanism to prevent production of reactive oxygen species by Escherichia coli respiratory complex I

et al. 2019

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Respiratory complex I plays a central role in cellular energy metabolism coupling NADH oxidation to proton translocation. In humans its dysfunction is associated with degenerative diseases. Here we report the structure of the electron input part of Aquifex aeolicus complex I at up to 1.8 Å resolution with bound substrates in the reduced and oxidized states. The redox states differ by the flip of a peptide bond close to the NADH binding site. The orientation of this peptide bond is determined by the reduction state of the nearby [Fe-S] cluster N1a. Fixation of the peptide bond by site-directed mutagenesis led to an inactivation of electron transfer and a decreased reactive oxygen species (ROS) production. We suggest the redox-gated peptide flip to represent a previously unrecognized molecular switch synchronizing NADH oxidation in response to the redox state of the complex as part of an intramolecular feed-back mechanism to prevent ROS production.

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Low cost, microcontroller based heating device for multi-wavelength differential scanning fluorimetry

Cited by 7 publications

References 22 publications

Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis

Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis

Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities

A mechanism to prevent production of reactive oxygen species by Escherichia coli respiratory complex I

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