title: Conformational dynamics in the F/G segment of CYP51 from Mycobacterium tuberculosis monitored by FRET.
abstract: A cysteine was introduced into the FG-loop (P187C) of CYP51 from Mycobacterium tuberculosis (MT) for selective labeling with BODIPY and fluorescence energy transfer (FRET) analysis. Förster radius for the BODIPY-heme pair was calculated assuming that the distance between the heme and Cys187 in solution corresponds to that in the crystal structure of ligand free MTCYP51. Interaction of MTCYP51 with azole inhibitors ketoconazole and fluconazole or the substrate analog estriol did not influence the fluorescence, but titration with the substrate lanosterol quenched BODIPY emission, the effect being proportional to the portion of substrate bound MTCYP51. The detected changes correspond to approximately 10A decrease in the calculated distance between BODIPY-Cys187 and the heme. The results confirm (1) functional importance of conformational motions in the MTCYP51 F/G segment and (2) applicability of FRET to monitor them in solution.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.
authors.forename: Galina I
authors.initials: GI
authors.lastname: Lepesheva
authors.forename: Matej
authors.initials: M
authors.lastname: Seliskar
authors.forename: Charles G
authors.initials: CG
authors.lastname: Knutson
authors.forename: Nina V
authors.initials: NV
authors.lastname: Stourman
authors.forename: Damjana
authors.initials: D
authors.lastname: Rozman
authors.forename: Michael R
authors.initials: MR
authors.lastname: Waterman
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D003198:Computer Simulation; D003461:Crystallography / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000648:ultrastructure*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System; C501935:cytochrome P-450 CYP51, Mycobacterium tuberculosis