13C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites.
The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a (13)CH(3)-reporter attached. This (13)C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site.
McCullough
Christopher R
CR
Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI 53201-1881, USA.
Pullela
Phani Kumar
PK
Im
Sang-Choul
SC
Waskell
Lucy
L
Sem
Daniel S
DS
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D000072467:Cytochrome P450 Family 2; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D016958:Pseudomonas putida / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009570:Nitriles; D011994:Recombinant Proteins; C008462:methylisonitrile; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D019475:Camphor 5-Monooxygenase