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

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Isom, DG; Vardy, E; Oas, TG; Hellinga, HW

Published Date

  • March 16, 2010

Published In

Volume / Issue

  • 107 / 11

Start / End Page

  • 4908 - 4913

PubMed ID

  • 20194783

Electronic International Standard Serial Number (EISSN)

  • 1091-6490

Digital Object Identifier (DOI)

  • 10.1073/pnas.0910421107

Language

  • eng

Conference Location

  • United States