Design and synthesis of a fluorescent reporter of protein kinase activity.
There is widespread interest in developing fluorescent reporters of protein kinase activity, species that can furnish a visual readout of both when and where intracellular kinases are activated in response to a stimulus. We have constructed and identified, via a combination of rational design, library synthesis, and screening, a difluorofluorescein-appended peptide-based species that responds to protein kinase C phosphorylation in a fluorescently sensitive fashion. The phosphorylation-induced divalent metal ion-mediated 265% enhancement in fluorescence proceeds with a V(max) of 8.5 micromol/min.mg and a K(m) of 20.5 microM.
Chen
Chien-An
CA
Department of Biochemistry, The Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Yeh
Ren-Hwa
RH
Lawrence
David S
DS
D003432:Cross-Linking Reagents / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010636:Phenols / Q000138:chemical synthesis / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D014966:Xanthenes / Q000138:chemical synthesis / Q000737:chemistry
D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D009842:Oligopeptides; D010636:Phenols; D014966:Xanthenes; D011494:Protein Kinases