A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity

Daniel G. Isom, Philippe R. Marguet, Terrence G. Oas, Homme W. Hellinga

Research output: Contribution to journalArticlepeer-review

18 Scopus citations


Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity, which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics. Proteins 2011.

Original languageEnglish (US)
Pages (from-to)1034-1047
Number of pages14
JournalProteins: Structure, Function and Bioinformatics
Issue number4
StatePublished - Apr 2011
Externally publishedYes


  • Conformational free energy
  • Dissociation constants binding affinity
  • Linkage analysis of protein stability
  • Protein folding kinetics
  • Protein thermodynamic stability
  • Quantitative cysteine reactivity
  • Ribose-binding protein
  • Staphylococcal nuclease
  • Thiol reactivity

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology


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