A method to measure the interaction of Rac/Cdc42 with their binding partners using fluorescence resonance energy transfer between mutants of green fluorescent protein

Citation
Dl. Graham et al., A method to measure the interaction of Rac/Cdc42 with their binding partners using fluorescence resonance energy transfer between mutants of green fluorescent protein, ANALYT BIOC, 296(2), 2001, pp. 208-217
Citations number
26
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ANALYTICAL BIOCHEMISTRY
ISSN journal
0003-2697 → ACNP
Volume
296
Issue
2
Year of publication
2001
Pages
208 - 217
Database
ISI
SICI code
0003-2697(20010915)296:2<208:AMTMTI>2.0.ZU;2-M
Abstract
Enhanced blue fluorescent protein (EBFP) and enhanced green fluorescent pro tein (EGFP) mutants of GFP in close proximity to one another can act as a f luorescence resonance energy transfer (FRET) pair. Unstructured amino acid linkers of varying length were inserted between EBFP and EGFP, revealing th at linkers even as long as 50 amino acids can be accommodated and still all ow FRET to occur. This led to the development of a novel biosensor for Rac/ Cdc42 binding to their effector proteins based on the insertion of amino ac ids 75-118 of p21-activated kinase (PAK) between the GFP mutants. We demons trate that this protein construct allows significant FRET between EBFP and EGFP and retains the ability to bind to Rac in its GTP-bound form with a bi nding affinity similar to the uncomplexed PAK fragment, and furthermore, on binding to Rac or Cdc42 a marked change in FRET takes place. This forms th e basis for a simple, sensitive, and rapid method to measure binding of Rac /Cdc42 to their effector proteins. Since the signal is dependent upon the i nteraction with active GTP-bound forms it acts as a biosensor for the activ ation of Rac/Cdc42. It has the potential for use in live cells and for iden tifying localization of Rac/Cdc42 within subcellular compartments. (C) 2001 Academic Press.