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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.

SUBMITTER: Lee HM

PROVIDER: S-EPMC3045470 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Construction of a photoactivatable profluorescent enzyme via propinquity labeling.

Lee Hsien-Ming HM Xu Weichen W Lawrence David S DS

Journal of the American Chemical Society 20110208 8

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). T ...[more]

PMID: 21302921

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