Use of fluorescence spectroscopy to study the regulation of small G proteins.
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.
Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
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D018797:Cell Cycle Proteins / Q000378:metabolism; D004926:Escherichia coli; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D013050:Spectrometry, Fluorescence / Q000379:methods*; D020764:cdc42 GTP-Binding Protein
D018797:Cell Cycle Proteins; D005456:Fluorescent Dyes; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein