Development of a fluorogenic sensor for activated Cdc42
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Author(s)
Goguen, Brenda N.
Loving, Galen S.
Imperiali, Barbara
Date Issued
April 2011
Journal
Bioorganic & Medicinal Chemistry Letters
Publisher
Elsevier
Citation
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.
Version
Author's final manuscript
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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
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Creative Commons Attribution-Noncommercial-NoDerivatives
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DOI of Published Version
https://doi.org/10.1016/j.bmcl.2011.04.051
