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

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Isom, DG; Marguet, PR; Oas, TG; Hellinga, HW

Published Date

  • April 2011

Published In

Volume / Issue

  • 79 / 4

Start / End Page

  • 1034 - 1047

PubMed ID

  • 21387407

Pubmed Central ID

  • 21387407

Electronic International Standard Serial Number (EISSN)

  • 1097-0134

Digital Object Identifier (DOI)

  • 10.1002/prot.22932

Language

  • eng

Conference Location

  • United States