Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity

Daniel Isom, Eyal Vardy, Terrence G. Oas, Homme W. Hellinga

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCR-derived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics.

Original languageEnglish (US)
Pages (from-to)4908-4913
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number11
DOIs
StatePublished - Mar 16 2010
Externally publishedYes

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Protein Stability
Cysteine
Ligands
Proteins
Protein Engineering
Genomics
Thermodynamics
Nucleic Acids
Equipment and Supplies

Keywords

  • Conformational stability
  • Ligand-binding affinity
  • Linkage analysis
  • Thermal stability
  • Thiol protection

ASJC Scopus subject areas

  • General

Cite this

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity. / Isom, Daniel; Vardy, Eyal; Oas, Terrence G.; Hellinga, Homme W.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 11, 16.03.2010, p. 4908-4913.

Research output: Contribution to journalArticle

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