title: Development of a fluorogenic sensor for activated Cdc42.
abstract: Cdc42, a member of the Rho GTPase family, is a fundamental regulator of the actin cytoskeleton during cell migration. To generate a sensor for Cdc42 activation, we employed a multi-pronged approach, utilizing cysteine labeling and expressed protein ligation, to incorporate the environment sensitive fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) into the GTPase binding domain of the WASP protein. These constructs bind only the active, GTP-bound conformation of Cdc42 to produce a fluorescence signal. Studies with a panel of five sensor analogs revealed a derivative that exhibits a 32-fold increase in fluorescence intensity in the presence of activated Cdc42 compared to incubation with the inactive GDP-bound form of the protein. We demonstrate that this sensor can be exploited to monitor Cdc42 nucleotide exchange and GTPase activity in a continuous, fluorescence assay.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Brenda N
authors.initials: BN
authors.lastname: Goguen
authors.forename: Galen S
authors.initials: GS
authors.lastname: Loving
authors.forename: Barbara
authors.initials: B
authors.lastname: Imperiali
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D020764:cdc42 GTP-Binding Protein