[The use of selective chemical modification of cytochrome P450scc (CYPXIA1) with fluorescein isothiocyanate for evaluation of intramolecular distances and conformational changes by fluorescent resonance energy transfer].
Clinical modification of cytochrome P450scc with fluoresceine isothiocyanate (FITC) results in selective incorporation of the fluorescent label into Lys338. According to affinity chromatography on immobilized adrenodoxin, fluorescently labeled cytochrome P450scc can interact with ferredoxin. Fluorescent labeling did not significantly affect the enzymatic activity of the hemoprotein at the stage of the first electron transfer. Distance between the heme and labeled Lys338 (2.81 nm) was determined using the efficiency of the fluorescent resonance energy transfer within the donor-acceptor pair (FITC-heme); also changes in this distance were measured during the transitions of cytochrome P450scc from high to low spin state and from oligomeric to monomeric form as well as after its conversion to cytochrome P420 by temperature or alkaline treatment and after complete denaturation of the protein globule in 6 M guanidine chloride. Thus, chemical modification of cytochrome P450scc by selective labeling with FITC is informative method which can be used to monitor conformational changes in the cytochrome P450scc molecule during enzymatic catalysis.
Lepesheva
G I
GI
Usanov
S A
SA
D000302:Adrenal Cortex / Q000201:enzymology; D000818:Animals; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004735:Energy Transfer; D005288:Ferredoxins / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D008928:Mitochondria / Q000201:enzymology; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D005288:Ferredoxins; D005456:Fluorescent Dyes; D002786:Cholesterol Side-Chain Cleavage Enzyme; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine
Ispol'zovanie selektivnoĭ khimicheskoĭ modifikatsii tsitokhroma P450scc (CYPXIA1) fluorestseinizotiotsianatom dlia otsenki vnutrimolekuliarnykh rasstoianiĭ i konformatsionnykh izmeneniĭ s pomoshch'iu rezonansnogo perenosa énergii fluorestsentsii.