Fluorescent probes for cytochrome p450 structural characterization and inhibitor screening |
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Authors: | Dunn Alexander R Hays Anna-Maria A Goodin David B Stout C David Chiu Richard Winkler Jay R Gray Harry B |
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Institution: | Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA. |
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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 F?rster 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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