Development of a fluorogenic sensor for activated Cdc42

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS

卷:21

Article

Imperiali, Barbara¹ 

[1] MIT, Dept Chem, Cambridge, MA 02139 USA

关键词: Biosensors;    Protein design;    Expressed protein ligation;    Cysteine labeling;    Solvatochromic fluorophores;   

DOI  :  10.1016/j.bmcl.2011.04.051

来源: Elsevier

【 摘 要 】

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. (c) 2011 Published by Elsevier Ltd.

【 授权许可】

Free