title: Construction of a photoactivatable profluorescent enzyme via propinquity labeling.
abstract: A strategy for the construction of a profluorescent caged enzyme is described. An active site-directed peptide-based affinity label was designed, synthesized, and employed to covalently label a nonactive site residue in the cAMP-dependent protein kinase. The modified kinase displays minimal catalytic activity and low fluorescence. Photolysis results in partial cleavage of the enzyme-bound affinity label, restoration of enzymatic activity (60-80%) and a strong fluorescent response (10-20 fold). The caged kinase displays analogous behavior in living cells, inducing a light-dependent loss of stress fibers that is characteristic of cAMP action. This strategy furnishes molecularly engineered enzymes that can be remotely controlled in time, space, and total activity.
authors.affiliation: Department of Chemistry, Division of Medicinal Chemistry and Natural Products, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Hsien-Ming
authors.initials: HM
authors.lastname: Lee
authors.forename: Weichen
authors.initials: W
authors.lastname: Xu
authors.forename: David S
authors.initials: DS
authors.lastname: Lawrence
mesh_terms: D055162:Biocatalysis / Q000528:radiation effects*; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation / Q000528:radiation effects; D005453:Fluorescence; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D055668:Photochemical Processes / Q000528:radiation effects*; D010782:Photolysis / Q000528:radiation effects; D015202:Protein Engineering; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D010455:Peptides; D017868:Cyclic AMP-Dependent Protein Kinases