Development of a fluorogenic sensor for activated Cdc42

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

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Fluorescent Dyes / chemistry*
  • Spectrometry, Fluorescence
  • cdc42 GTP-Binding Protein / chemistry*

Substances

  • Fluorescent Dyes
  • cdc42 GTP-Binding Protein