Conformational dynamics in the F/G segment of CYP51 from Mycobacterium tuberculosis monitored by FRET

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. Forster radius for the BODIPY-heme pair was calculated assuming that the distance between the heme and Cysl87 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 similar to 10 angstrom decrease in the calculated distance between BODIPY-Cysl87 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. (c) 2007 Elsevier Inc. All rights reserved.