Bicelle-enabled structural studies on a membrane-associated cytochrome B5 by solid-state MAS NMR spectroscopy.

Various important functional roles played by membrane proteins that are related to a number of diseases, will be better understood once their high-resolution structures and dynamics are revealed. Although structural studies on membrane proteins have been a great challenge to most biophysical techniques, recent NMR spectroscopic studies were able to overcome many of the difficulties of structural elucidations for a number of proteins. However, structural studies of membrane proteins still remain a great challenge, mainly because of the difficulty in finding a well-behaved model membrane. The use of multi-lamellar vesicles containing a transmembrane protein could enable the application of solidstate NMR spectroscopic techniques, but they are not usually suitable, as membrane proteins containing large soluble domains may not fold well to result in high-resolution spectra. Obtaining high-resolution spectra is a mandatory first step in solving the protein structure using NMR spectroscopy. In this study we demonstrate that bicelles are suitable to overcome these difficulties and enable the use of MAS (magic-anglespinning) solid-state NMR spectroscopic experiments for the structural studies on a large soluble domain containing membrane-bound protein such as cytochrome b5 (cytb5). Cytb5 modifies the catalytic activity and takes part in the catalytic reactions of cytochrome P450, however, its highresolution structure remains unknown. In addition, cytb5 is involved as an electron transfer component in a number of oxidative reactions in biological tissues which include the biosynthesis of fatty acids and steroids. Isotropic N chemical shift spectra of a uniformly Nlabeled full-length 16.7 kDa rabbit cytb5 embedded in bicelles consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) and multilamellar vesicles (MLVs) using DMPC are given in Figure 1. RAMP-CP (ramped cross polarization) [4] and RINEPT (refocused intensive nuclei enhanced by polarization transfer) [5] sequences were used to transfer