Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities.

Scientific Reports
Nan BaiJohn Karanicolas

Abstract

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 (Kd). 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 fold...Continue Reading

References

Nov 1, 1987·Biopolymers·W J Becktel, J A Schellman
Oct 1, 1995·Protein Science : a Publication of the Protein Society·J K MyersJ M Scholtz
Jan 1, 1997·Protein Science : a Publication of the Protein Society·V Novokhatny, K Ingham
Jan 15, 2002·Journal of Biomolecular Screening·M W PantolianoF R Salemme
Aug 5, 1997·Chemical Reviews·Andrew D. Robertson, Kenneth P. Murphy
Jun 10, 2003·The Journal of Biological Chemistry·Patrick G Telmer, Brian H Shilton
Aug 11, 2004·Analytical Biochemistry·Mei-Chu LoGeorge Ellestad
Aug 25, 2004·Analytical Biochemistry·Zaneta Nikolovska-ColeskaShaomeng Wang
Jan 7, 2005·The Journal of Biological Chemistry·Theodore E CarverMatthew J Todd
Jan 18, 2005·International Journal of Pharmaceutics·Wei Wang
Nov 1, 2005·Drug Discovery Today·Geoffrey A Holdgate, Walter H J Ward
Mar 23, 2006·Acta Crystallographica. Section D, Biological Crystallography·Andrew P YehMichael H B Stowell
Sep 12, 2006·Analytical Biochemistry·Ulrika B EricssonPär Nordlund
Dec 14, 2007·Proceedings of the National Academy of Sciences of the United States of America·Oleg FedorovStefan Knapp
May 2, 2008·Journal of Biomolecular Screening·Guillermo A SenisterraMasoud Vedadi
Jul 5, 2008·Biophysical Journal·Piotras CimmpermanDaumantas Matulis
May 28, 2009·Journal of Biomolecular Screening·Gregory J CrowtherWesley C van Voorhis
Jun 11, 2010·Applied Microbiology and Biotechnology·Iris H WalkerPaul D Riggs
Sep 28, 2010·Nature·Panagis FilippakopoulosJames E Bradner
Dec 9, 2010·Analytical Biochemistry·Kumaran MahendrarajahEwan R G Main
Mar 5, 2011·Methods in Enzymology·James K Kranz, Celine Schalk-Hihi
Oct 27, 2012·Analytical Biochemistry·Michèle N SchulzRoderick E Hubbard
May 16, 2013·Journal of Medicinal Chemistry·E Andreas LarssonPär Nordlund
May 29, 2013·ACS Combinatorial Science·Shane A Seabrook, Janet Newman
Sep 3, 2013·Analytical Biochemistry·Arne SchönErnesto Freire
Feb 1, 2014·Nature Reviews. Drug Discovery·Andrew L HopkinsCharles H Reynolds
Feb 1, 2014·Chembiochem : a European Journal of Chemical Biology·Ran DaiBarry C Finzel
Oct 7, 2014·Journal of Visualized Experiments : JoVE·Mirella VivoliNicholas J Harmer
Nov 12, 2014·Journal of Biomolecular Screening·Anita Niedziela-MajkaRoman Sakowicz
Feb 3, 2015·Current Protocols in Protein Science·Kathy Huynh, Carrie L Partch

❮ Previous
Next ❯

Citations

Aug 11, 2020·Future Medicinal Chemistry·Thamires Quadros FroesMarcelo Santos Castilho
Sep 29, 2020·SLAS Discovery·Rajdeep S VirdiDavid N Frick
May 31, 2019·Expert Opinion on Drug Discovery·Visvaldas KairysEgidijus Kazlauskas
Jan 25, 2021·Biotechnology for Biofuels·James LiHarry Brumer
Mar 3, 2021·Science Immunology·Jacqueline DouglassShibin Zhou
May 7, 2021·Scientific Reports·Stephan NieblingMaría García-Alai
Jun 4, 2021·Journal of Enzyme Inhibition and Medicinal Chemistry·Thamires Quadros FroesMarcelo Santos Castilho
Jul 3, 2021·International Journal of Molecular Sciences·Alessandra CamarcaNadia Raffaelli
Jun 15, 2021·Oxidative Medicine and Cellular Longevity·Md Faruq HossainChristopher Horst Lillig
Jul 25, 2021·Proceedings of the National Academy of Sciences of the United States of America·Amir Arellano-SaabShelley Lumba
Aug 6, 2021·The Journal of Physical Chemistry Letters·Gil I OlgenblumDaniel Harries
Sep 14, 2021·Protein Science : a Publication of the Protein Society·Taylor MoncriefAndres F Oberhauser
Sep 29, 2021·Chemical Society Reviews·Louise J WalportJoel P Mackay
Oct 5, 2021·Acta Crystallographica. Section D, Structural Biology·Osvaldo BurasteroMaria M Garcia Alai
Oct 28, 2021·Journal of Medicinal Chemistry·Parima UdompholkulMaurizio Pellecchia
Aug 15, 2021·The Journal of Pharmacology and Experimental Therapeutics·Srinidhi JayaramanVardan T Karamyan
Dec 2, 2021·Plant Physiology·Randa A ZarbanSalim Al-Babili

❮ Previous
Next ❯

Methods Mentioned

BETA
protein folding
isothermal titration calorimetry
circular dichroism
Protein Assay
PCR

Software Mentioned

GraphPad
Prism6
Prism
SourceForge
mathop

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.