The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

Niesen, F.; Berglund, H.; Vedadi, Masoud

doi:10.1038/nprot.2007.321

Cited by 2,167 publications

(2,257 citation statements)

References 15 publications

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“…The thermal shift assay 21 , also known as differential scanning fluorimetry 22 and ThermoFluor® 23 , is a high-throughput screening method for hit selection and the determination of the protein-ligand binding constants used in the pharmaceutical industry 24 . This biophysical technique can be applied to any protein-ligand non-covalent binding reaction, independent of whether the ligand stabilizes or destabilizes the protein upon binding 25 .…”

Section: Introductionmentioning

confidence: 99%

Thermodynamics of radicicol binding to human Hsp90 alpha and beta isoforms

Zubrienė¹,

Gutkowska²,

Matulienė³

et al. 2010

Biophysical Chemistry

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Radicicol is a natural antibiotic that specifically inhibits chaperone Hsp90 activity and binds to its active site with nanomolar affinity. Radicicol has been widely used as a lead compound to generate synthetic analogs with reduced toxicity and increased stability that could be employed clinically. Here we present a detailed thermodynamic description of radicicol binding to human Hsp90 and yeast Hsc82 studied by isothermal titration calorimetry and thermal shift assay.Titrations as a function of pH showed a linked protonation event upon radicicol binding. The intrinsic binding constant and the thermodynamic parameters (including the enthalpy, entropy, and heat capacity) were determined for yeast Hsc82, and human alpha and beta Hsp90. Recent experimental evidence in literature shows that yeast Hsc82 has significant differences from human Hsp90 isozymes. Here we support this by demonstrating differences in radicicol binding thermodynamics to these proteins. The intrinsic enthalpy of radicicol binding to Hsc82 was -46.7 kJ/mol, to Hsp90alpha -70.7 kJ/mol, and to Hsp90beta was -66.8 kJ/mol. The enthalpies of binding were significantly different, while the intrinsic dissociation constants were quite similar,

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Section: Introductionmentioning

confidence: 99%

Thermodynamics of radicicol binding to human Hsp90 alpha and beta isoforms

Zubrienė¹,

Gutkowska²,

Matulienė³

et al. 2010

Biophysical Chemistry

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“…This network stabilized a particular active site conformation that the authors suggested promoted greater global stability. [7] Here we probe the effects of IFG-binding in solution on GCase global stability by differential scanning fluorimetry [18][19][20][21][22] and on local dynamics by amide hydrogen/ deuterium exchange coupled with proteolysis and mass spectrometry (H/D-Ex). [23][24][25][26][27][28] The ability to partially restore intracellular trafficking and lysosomal GCase localization of mutant forms of GCase was then inferred from activity assays performed on N370S/N370S and F213I/L444P patient fibro-blasts.…”

mentioning

confidence: 99%

Isofagomine Induced Stabilization of Glucocerebrosidase

Kornhaber¹,

Tropak

Maegawa

et al. 2008

ChemBioChem

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Structurally destabilizing mutations in acid β-glucosidase (GCase) can result in Gaucher disease (GD). The iminosugar isofagomine (IFG), a competitive inhibitor and a potential pharmacological chaperone of GCase, is currently undergoing clinical evaluation for the treatment of GD. An X-ray crystallographic study of the GCase-IFG complex revealed a hydrogen bonding network between IFG and certain active site residues. It was suggested that this network may translate into greater global stability. Here it is demonstrated that IFG does increase the global stability of wild-type GCase, shifting its melting curve by ~15 °C and that it enhances mutant GCase activity in pretreated N370S/N370S and F213I/L444P patient fibroblasts. Additionally, amide hydrogen/ deuterium exchange mass spectroscopy (H/D-Ex) was employed to identify regions within GCase that undergo stabilization upon IFG-binding. H/D-Ex data indicate that the binding of IFG not only restricts the local protein dynamics of the active site, but also propagates this effect into surrounding regions.

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“…The melting temperature (Tm) corresponds to the minimum of the negative derivative curves and is an indicator of protein stability. It generally increases when the protein binds a ligand [13]. some peptide bonds for protease digestion.…”

Section: Characterization Of Ysxc Mutantsmentioning

confidence: 99%

“…The Thermal Shift assay was used to study binding interactions between YsxC and nucleotides. The ligand generally stabilizes the protein and the Tm of the complex is higher than that of the apo protein [13].…”

Section: Introductionmentioning

confidence: 99%

The C‐terminal α‐helix of YsxC is essential for its binding to 50S ribosome and rRNAs

2015

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a b s t r a c tYsxC is an essential P-loop GTPase that interacts with the 50S subunit of the ribosome. The putative implication in ribosome binding of two basic clusters of YsxC, a conserved positively charged patch including R31, R116, H117 and K146 lying adjacent to the nucleotide-binding site, and the C-terminal alpha helix, was investigated. C-terminal truncation variants of YsxC were unable to bind to both ribosome and rRNAs, whereas mutations in the other cluster did not affect YsxC binding. Our results indicate that the basic C-terminal region of YsxC is required for its binding to the 50S ribosomal subunit.

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The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

Cited by 2,167 publications

References 15 publications

Thermodynamics of radicicol binding to human Hsp90 alpha and beta isoforms

Thermodynamics of radicicol binding to human Hsp90 alpha and beta isoforms

Isofagomine Induced Stabilization of Glucocerebrosidase

The C‐terminal α‐helix of YsxC is essential for its binding to 50S ribosome and rRNAs

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