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Bioorg Med Chem Lett. 2011 Sep 1;21(17):5058-61. doi: 10.1016/j.bmcl.2011.04.051. Epub 2011 Apr 20.

Development of a fluorogenic sensor for activated Cdc42.

Author information

1
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

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.

PMID:
21549598
PMCID:
PMC3156282
DOI:
10.1016/j.bmcl.2011.04.051
[Indexed for MEDLINE]
Free PMC Article

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