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Fluorescent probes for cytochrome p450 structural characterization and inhibitor screening.

Alexander R Dunn·Anna-Maria A Hays·David B Goodin·C David Stout·Richard Chiu·Jay R Winkler·Harry B Gray
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Abstract

We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by Förster energy transfer upon binding (Kd = 0.83 muM) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (Kd approximately 0.02 muM) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 A resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the B', F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors.

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AdamantaneBinding, CompetitiveCamphorCamphor 5-MonooxygenaseDansyl CompoundsFluorescent DyesKineticsModels, MolecularSpectrometry, FluorescenceJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

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Alexander R Dunn, Anna-Maria A Hays, David B Goodin, C David Stout, Richard Chiu, Jay R Winkler, Harry B Gray. 2002-09-04. Fluorescent probes for cytochrome p450 structural characterization and inhibitor screening.. https://doi.org/10.1021/ja0271678

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