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.
Nomanbhoy
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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