Determination of RNase A/2′-Cytidine Monophosphate Binding Affinity and Enthalpy by a Global Fit of Thermal Unfolding Curves

Jones, Cecil L.; Fish, F.; Muccio, Donald D.

doi:10.1006/abio.2001.5529

Cited by 9 publications

(8 citation statements)

References 23 publications

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“…In the literature, K D values have been determined by various methods in solution (ITC, DSC, CD, SPR, fluorescence or ligand radio-labeling). Solutionmeasured K D values of 0.5 to 1.6 μM can be found in the literature for the RNase-CMP system [32][33][34]. These were found to be close to that of RNase-CTP as determined by another MS study [31].…”

Section: Comparison Of K D Values Determined Using Gpd Correction Witsupporting

confidence: 78%

Improved Accuracy of Low Affinity Protein–Ligand Equilibrium Dissociation Constants Directly Determined by Electrospray Ionization Mass Spectrometry

Jaquillard

Saab

Schoentgen

et al. 2012

J. Am. Soc. Mass Spectrom.

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There is continued interest in the determination by ESI-MS of equilibrium dissociation constants (K(D)) that accurately reflect the affinity of a protein-ligand complex in solution. Issues in the measurement of K(D) are compounded in the case of low affinity complexes. Here we present a K(D) measurement method and corresponding mathematical model dealing with both gas-phase dissociation (GPD) and aggregation. To this end, a rational mathematical correction of GPD (f(sat)) is combined with the development of an experimental protocol to deal with gas-phase aggregation. A guide to apply the method to noncovalent protein-ligand systems according to their kinetic behavior is provided. The approach is validated by comparing the K(D) values determined by this method with in-solution K(D) literature values. The influence of the type of molecular interactions and instrumental setup on f(sat) is examined as a first step towards a fine dissection of factors affecting GPD. The method can be reliably applied to a wide array of low affinity systems without the need for a reference ligand or protein.

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Section: Comparison Of K D Values Determined Using Gpd Correction Witsupporting

confidence: 78%

Improved Accuracy of Low Affinity Protein–Ligand Equilibrium Dissociation Constants Directly Determined by Electrospray Ionization Mass Spectrometry

Jaquillard

Saab

Schoentgen

et al. 2012

J. Am. Soc. Mass Spectrom.

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“…By using an equimolar concentration of RNase A, 2´-CMP and CTP, the dissociation constants could be determined using the two equations, Kd suggesting that nonspecific binding in this assay was minimized. Furthermore, these results obtained from chip-based nano-ESI/MS are comparable with those published from other conventional techniques such as calorimetry and circular dichroism [104][105][106]. It was demonstrated that the chip-based approach is in relatively good agreement with previously reported values.…”

Section: Noncovalent Binding Interactions and Drug Screeningsupporting

confidence: 91%

Chip-based nanoelectrospray mass spectrometry for protein characterization

Zhang¹,

Pelt²

2004

Expert Review of Proteomics

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In the last several years, significant progress has been made in the development of microfluidic-based analytical technologies for proteomic and drug discovery applications. Chip-based nanoelectrospray coupled to a mass spectrometer detector is one of the recently developed analytical microscale technologies. This technology offers unique advantages for automated nanoelectrospray including reduced sample consumption, improved detection sensitivity and enhanced data quality for proteomic studies. This review presents an overview and introduction of recent developments in chip devices coupled to electrospray mass spectrometers including the development of the automated nanoelectrospray ionization chip device for protein characterization. Applications using automated chip-based nanoelectrospray ionization technology in proteomic and bioanalytical studies are also extensively reviewed in the fields of high-throughput protein identification, protein post-translational modification studies, top-down proteomics, biomarker screening by pattern recognition, noncovalent protein-ligand binding for drug discovery and lipid analysis. Additionally, future trends in chip-based nanoelectrospray technology are discussed.

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“…In addition to DSF/ThermoFluor, analogous data can be collected using other experimental modalities: most notably, probing the protein directly via intrinsic tryptophan fluorescence or circular dichroism (CD) spectroscopy. Regardless of the method by which protein unfolding is monitored, however, the analysis is the same; and indeed, the same thermodynamic models described earlier in the context of DSF have also been applied to thermal unfolding probed via CD 65,70,71 . Unsurprisingly, the challenges associated with applying thermodynamic models to directly quantify ligand binding at different temperatures apply to these other experimental formats as well 72 .…”

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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Determination of RNase A/2′-Cytidine Monophosphate Binding Affinity and Enthalpy by a Global Fit of Thermal Unfolding Curves

Cited by 9 publications

References 23 publications

Improved Accuracy of Low Affinity Protein–Ligand Equilibrium Dissociation Constants Directly Determined by Electrospray Ionization Mass Spectrometry

Improved Accuracy of Low Affinity Protein–Ligand Equilibrium Dissociation Constants Directly Determined by Electrospray Ionization Mass Spectrometry

Chip-based nanoelectrospray mass spectrometry for protein characterization

Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities

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