Dynamic Interaction Between Membrane-Bound Full-Length Cytochrome P450 and Cytochrome b5 Observed by Solid-State NMR Spectroscopy

Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055.
Scientific Reports (Impact Factor: 5.58). 08/2013; 3:2538. DOI: 10.1038/srep02538
Source: PubMed


Microsomal monoxygenase enzymes of the cytochrome-P450 family are found in all biological kingdoms, and play a central role in the breakdown of metabolic as well as xenobiotic, toxic and 70% of the drugs in clinical use. Full-length cytochrome-b5 has been shown to be important for the catalytic activity of cytochrome-P450. Despite the significance in understanding the interactions between these two membrane-associated proteins, only limited high-resolution structural information on the full-length cytochrome-P450 and the cytochromes-b5-P450 complex is available. Here, we report a structural study on a functional ~72-kDa cytochromes-b5-P450 complex embedded in magnetically-aligned bicelles without having to freeze the sample. Functional and solid-state NMR (Nuclear Magnetic Resonance) data reveal interactions between the proteins in fluid lamellar phase bilayers. In addition, our data infer that the backbone structure and geometry of the transmembrane domain of cytochrome-b5 is not significantly altered due to its interaction with cytochrome-P450, whereas the mobility of cytochrome-b5 is considerably reduced.

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Available from: Ayyalusamy Ramamoorthy, Mar 25, 2014
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    • "INEPT and refocused-INEPT (RINEPT) pulse sequences have been frequently utilized in the development of numerous sophisticated RF pulse sequences for solution NMR studies [53], and their theoretical analyses are well understood [54] [55]. RINEPT experiments are also utilized in solid-state NMR studies on oriented lipid bilayers containing a membrane protein [56] [57]. The presence of strong 1 H– 1 H dipolar couplings in rigid solids often results in the failure of RINEPT polarization transfer from protons due to the fast decay of proton transverse magnetization, which necessitates the use of a combination of a homonuclear dipolar decoupling sequence and MAS [10] [58]. "
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