Ligand-induced conformational heterogeneity of cytochrome P450 CYP119 identified by 2D NMR spectroscopy with the unnatural amino acid (13)C-p-methoxyphenylalanine.
Conformational dynamics are thought to play an important role in ligand binding and catalysis by cytochrome P450 enzymes, but few techniques exist to examine them in molecular detail. Using a unique isotopic labeling strategy, we have site specifically inserted a (13)C-labeled unnatural amino acid residue, (13)C-p-methoxyphenylalanine (MeOF), into two different locations in the substrate binding region of the thermophilic cytochrome P450 enzyme CYP119. Surprisingly, in both cases the resonance signal from the ligand-free protein is represented by a doublet in the (1)H,(13)C-HSQC spectrum. Upon binding of 4-phenylimidazole, the signals from the initial resonances are reduced in favor of a single new resonance, in the case of the F162MeOF mutant, or two new resonances, in the case of the F153MeOF mutant. This represents the first direct physical evidence for the ligand-dependent existence of multiple P450 conformers simultaneously in solution. This general approach may be used to further illuminate the role that conformational dynamics plays in the complex enzymatic phenomena exhibited by P450 enzymes.
Lampe
Jed N
JN
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158-2517, USA.
Floor
Stephen N
SN
Gross
John D
JD
Nishida
Clinton R
CR
Jiang
Yongying
Y
Trnka
Michael J
MJ
Ortiz de Montellano
Paul R
PR
D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008781:Methyltyrosines / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation
D000596:Amino Acids; D019843:Archaeal Proteins; D008024:Ligands; D008781:Methyltyrosines; C002350:O-methyltyrosine; D003577:Cytochrome P-450 Enzyme System; C408874:CYP119 protein, Sulfolobus solfataricus