<i>pytc</i>: Open-Source Python Software for Global Analyses of Isothermal Titration Calorimetry Data

H, Duvvuri; Wheeler, Lucas C.; Harms, Michael J.

doi:10.1021/acs.biochem.7b01264

Cited by 32 publications

(67 citation statements)

References 26 publications

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“…pytc was used to perform model fitting and parameter estimation. Two thermodynamic models were devised, informed by previous structural and biophysical data .…”

Section: Methodsmentioning

confidence: 99%

“…[20,[27][28][29] Bayesian methods provide a robust approach to this problem and have been deployed for ITC analysis in the form of an open source software called pytc. [30,31] Recently, we used Bayesian fitting in pytc to determine the thermodynamic parameters of the bidentate ligand (two-site) model for the PAF -sclx 8 complex. [22] Here, we have characterized the cytochrome c -sclx 8 system in detail by using ITC and thermodynamic modelling that incorporates ligand binding and oligomerization.…”

Section: Introductionmentioning

confidence: 99%

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A Thermodynamic Model of Auto‐regulated Protein Assembly by a Supramolecular Scaffold

Rennie

Crowley

2019

ChemPhysChem

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Ligand‐mediated regulation of protein assembly occurs frequently in different cellular contexts. Auto‐regulated assembly, where a ligand acts as its own competitive inhibitor, provides a mechanism for exquisite control of assembly. Unlike simple protein‐ligand systems a quantification of the binding thermodynamics is not straightforward. Here, we characterize the interactions of a recently identified model system in which the oligomerization of cytochrome c is controlled by sulfonato‐calix[8]arene, an anionic supramolecular scaffold. Isothermal titration calorimetry and thermodynamic modelling, in combination with Bayesian fitting, were used to quantify the ligand binding and assembly equilibria for this system. The approach and variations of this model may prove useful for the analysis of auto‐regulated protein assembly in general.

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“…pytc was used to perform model fitting and parameter estimation. Two thermodynamic models were devised, informed by previous structural and biophysical data .…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Thermodynamic Model of Auto‐regulated Protein Assembly by a Supramolecular Scaffold

Rennie

Crowley

2019

ChemPhysChem

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“…Exothermic reactions are depicted as negative values. Integrated peak data was fit with a Bayesian model to calculate the Kd (in bp DNA) with 95% confidence intervals (values shown in parentheses)(Duvvuri et al, 2018). (B) Biofilm phenazine concentrations for WT biofilms treated with or without DNase I in the underlying agar for 24hrs (n = 3 per condition).…”

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confidence: 99%

Extracellular DNA promotes efficient extracellular electron transfer by pyocyanin in Pseudomonas aeruginosa biofilms

Saunders

Tse

Yates

et al. 2019

Preprint

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DKN=lead contact). 2 SUMMARY:Extracellular electron transfer (EET), the process whereby cells access electron acceptors or donors that reside many cell lengths away, enables metabolic activity by microorganisms, particularly under oxidant-limited conditions that occur in multicellular bacterial biofilms. Although different mechanisms underpin this process in select organisms, a widespread strategy involves extracellular electron shuttles, redox-active metabolites that are secreted and recycled by diverse bacteria. How these shuttles catalyze electron transfer within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazine electron shuttles mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms, which are important in nature and disease. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by binding to eDNA. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and phenazines can participate directly in redox reactions through DNA; the biofilm eDNA can also support rapid electron transfer between redox active intercalators. Electrochemical measurements of biofilms indicate that retained PYO supports an efficient redox cycle with rapid EET and slow loss from the biofilm. Together, these results establish that eDNA facilitates phenazine metabolic processes in P. aeruginosa biofilms, suggesting a model for how extracellular electron shuttles achieve retention and efficient EET in biofilms. 3 INTRODUCTION:

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“…C. A single-site binding model fit to the ∆N-Zn data. Lines show results sampled from 10,000 Monte Carlo samples of possible fits using pytc [26]. Values are lower-and upper-bounds for 95% credibility interval.…”

Section: Fig 3 Mutagenesis Of S100a9 Disrupts Zn 2+ Binding and Zn 2mentioning

confidence: 99%

“…Integrations were performed with MicroCal Software. We extracted thermodynamic parameters for Zn 2+ binding to ∆N-Zn by fitting a single-site binding model as implemented in pytc [26]. We used the last four points of the titration to account for the heat of dilution.…”

Section: Plasmids and Recombinant Protein Preparation And Characterizmentioning

confidence: 99%

Zinc-independent activation of Toll-like receptor 4 by S100A9

Shi

Harms

2019

Preprint

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The homodimer formed by the protein S100A9 induces inflammation through Toll-like receptor 4 (TLR4), playing critical roles in both healthy and pathological innate immune responses. The molecular mechanism by which S100A9 activates TLR4 remains unknown. Previously, the interaction between purified S100A9 and TLR4 was shown to depend on Zn 2+ ; however, the Zn 2+ binding site(s) on S100A9 were not identified. Here, we investigated the role of Zn 2+ binding in the pro-inflammatory activity of S100A9. We found that the S100A9 homodimer was prone to reversible, Zn 2+ -dependent aggregation in vitro. Using a combination of site-directed mutagenesis and Isothermal Titration Calorimetry (ITC), we identified multiple residues that contribute to Zn 2+ binding in S100A9. We then used mutagenesis to construct a version of S100A9 with no detectable Zn 2+ binding by either ITC or Inductively Coupled Plasma-Mass Spectrometry. This protein did not exhibit aggregation upon addition of saturating Zn 2+ . Further, despite the lack of Zn 2+ -binding, this protein was capable of activating TLR4 in a cell-based functional assay. We then modified the functional assay so the Zn 2+ concentration was exceedingly low relative to the concentration of S100A9 added. Again, S100A9 was able to activate TLR4. This reveals that, despite the ability of S100A9 to bind Zn 2+ , S100A9 does not require Zn 2+ to activate TLR4. Our work represents an important step in clarifying the nature of the interaction between S100A9 and TLR4.

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pytc: Open-Source Python Software for Global Analyses of Isothermal Titration Calorimetry Data

Cited by 32 publications

References 26 publications

A Thermodynamic Model of Auto‐regulated Protein Assembly by a Supramolecular Scaffold

A Thermodynamic Model of Auto‐regulated Protein Assembly by a Supramolecular Scaffold

Extracellular DNA promotes efficient extracellular electron transfer by pyocyanin in Pseudomonas aeruginosa biofilms

Zinc-independent activation of Toll-like receptor 4 by S100A9

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