Development of a fluorogenic sensor for activated Cdc42
Author(s)Goguen, Brenda N.; Loving, Galen S.; Imperiali, Barbara
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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.
DepartmentMassachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Chemistry
Bioorganic & Medicinal Chemistry Letters
Goguen, Brenda N., Galen S. Loving, and Barbara Imperiali. “Development of a Fluorogenic Sensor for Activated Cdc42.” Bioorganic & Medicinal Chemistry Letters 21, no. 17 (September 2011): 5058–5061.
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