High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

Department of Cell and Developmental Biology, University of Illinois, Urbana, IL 61801, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 01/2008; 1768(12):3061-70. DOI: 10.1016/j.bbamem.2007.09.009
Source: PubMed


Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, Escherichia coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled His4CYP98A3 is expressed at yields of 2-4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated His4CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins.

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    • "Cytochromes P450, which metabolize approximately 75% of the pharmaceutics in clinical use today, are monooxygenases that activate the stable carbon hydrogen bond of alkanes, commonly referred to as Mother Nature's blowtorch3536373839404142. The cytochromes P450 family is found in all kingdoms of life and involved in a wide variety of enzymatic reactions in living organisms, such as drug metabolism, and the synthesis of steroids and lipids. "
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    ABSTRACT: Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochromeP4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.
    Scientific Reports 08/2013; 3:2556. DOI:10.1038/srep02556 · 5.58 Impact Factor
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    • "Several members of dipteran and lepidopteran CYP6 radiations are involved in resistance to a broad range of insecticides (OPs, synthetic pyrethroids, DDT and neonicotinoids; Carino et al., 1994; Liu & Scott, 1996; Daborn et al., 2002; Li et al., 2007; Muller et al., 2008) and/or the detoxification of host plant allelochemicals in the gut (Danielson et al., 1997; Mittapalli et al., 2005; Wen et al., 2005; Mao et al., 2006, 2007; Li et al., 2007; Rupasinghe et al., 2007). Less is known of the functions of the CYP9s but the limited evidence available also implicates them in the detoxification of insecticides and other allelochemicals (Stevens et al., 2000; Poupardin et al., 2008), with some evidence also for involvement in semiochemical metabolism (Maibeche-Coisne et al., 2005). "
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