Use of fluorescence spectroscopy to study the regulation of small G proteins

Methods Mol Biol. 1998:84:237-47. doi: 10.1385/0-89603-488-7:237.

Abstract

At present, there are a number of questions concerning the abilities of different regulators (GEFs, GAPs) and target molecules to bind to Cdc42Hs and related GTP-binding proteins. The ability to label Cdc42Hs with an extrinsic-reporter group (sNBD) with a 1:1 stoichiometry of probe incorporation per protein molecule, and without any loss of functional activity, provides a powerful reagent for quantitative assays of regulatory protein and target binding. The expectation is that the sNBD-labeled Cdc42Hs will be useful in definitively determining whether GAPs and individual targets compete with one another for binding to Cdc42Hs, or if multiple-target molecules can complex simultaneously with a single GTP-binding protein. Given the success in labeling Cdc42Hs with an extrinsic-reporter group, it seems likely that similar labeling approaches would be successful with other members of the family, such as the Rac and Rho proteins.

MeSH terms

  • Cell Cycle Proteins / metabolism
  • Escherichia coli
  • Fluorescent Dyes
  • GTP-Binding Proteins / metabolism*
  • Guanosine Triphosphate / metabolism
  • Humans
  • Spectrometry, Fluorescence / methods*
  • cdc42 GTP-Binding Protein

Substances

  • Cell Cycle Proteins
  • Fluorescent Dyes
  • Guanosine Triphosphate
  • GTP-Binding Proteins
  • cdc42 GTP-Binding Protein