Fluorescent probes for cytochrome p450 structural characterization and inhibitor screening.
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.
Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA.
Alexander R
AR
Dunn
Anna-Maria A
AM
Hays
David B
DB
Goodin
C David
CD
Stout
Richard
R
Chiu
Jay R
JR
Winkler
Harry B
HB
Gray
D000218:Adamantane / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001667:Binding, Competitive; D002164:Camphor / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D003619:Dansyl Compounds / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D013050:Spectrometry, Fluorescence
C467203:D-4-Ad compound; C467204:D-8-Ad compound; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D002164:Camphor; D019475:Camphor 5-Monooxygenase; D000218:Adamantane