Biological cell membranes are vital boundaries that separate the intracellular elements from the extracellular environments, and the membrane proteins in such borders are fundamental regulators of a number of essential cellular and physiological phenomena in life, including signal transductions, electron transport chains, and photosynthesis. Furthermore, membrane-associated proteins comprise more than 30% of the human genome and 50% of known drug targets. In order to understand the roles of these proteins in biological activities, and to develop medical treatments of related diseases, it is critical to establish biophysical methods to investigate the functional form of membrane protein structures at atomic-level1,2,3,4,5,6,7,8,9,10
. However, membrane protein structure determination is an extremely challenging task due to the lack of stability of the protein outside the native membrane environment. For this reason, development of novel methods to stabilize the native structure of membrane proteins is essential for driving high-resolution structural studies using biophysical techniques such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography11,12,13,14,15,16,17,18,19,20,21,22
. Bicelles are increasingly used as model membranes for the studies of biomolecules with various biophysical methods, including solid-state NMR, solution NMR, X-ray crystallography, EPR (Electron Paramagnetic Resonance), CD (Circular Dichroism), fluorescence, IR (Infra Red), Raman, UV-Vis spectroscopy, ITC (Isothermal Titration Calorimetry), DSC (Differential Scanning Calorimetry), and microscopy, as their planar domain provides an excellent environment for the study of membrane-associated proteins in transparent fluid solutions, which prevent light scattering11,12,14,15,23,24,25,26,27
. Bicelles are typically made from a mixture of long-chain phospholipid and short-chain phospholipid/detergent (e.g. DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine), CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), DPC (dodecylphosphocholine), or Triton X-100). The size of bicelles can be controlled by the lipid to detergent molar ratio, called q
ratio ( = [lipid]/[detergent]), and also by the hydration level. Large bicelles (q
ratio > 2.5), that spontaneously align in a magnetic field above the phase transition temperature, are highly valuable to measure distance and orientational constraints from embedded membrane proteins using static solid-state NMR experiments. Similarly, anisotropic NMR interactions, such as residual dipolar couplings (RDCs) and residual chemical shift anisotropy of soluble proteins, can be obtained using solution NMR experiments on bicelles with low concentrations. Small and fast-tumbling bicelles (q
ratio < 1.5) are an excellent reconstitution medium for solution NMR studies of membrane proteins. Recent studies have shown that bicelles retaining the function of proteins are also useful to study membrane-bound protein-protein complexes. While many useful bicelle compositions have been reported in the literature, bicelles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) are most frequently used. These DMPC/DHPC bicelles have a narrow temperature range, between 25 and 45°C, for magnetic alignment and have been well utilized in static solid-state NMR experiments on membrane proteins under this temperature range. This high temperature requirement of magnetically-aligned bicelles is problematic for heat-sensitive biomolecules, such as cytochromes P450; in addition, solid-state NMR experiments employ high power RF pulses that can induce sample heating. Therefore, there is considerable interest in developing fluid model membranes that can align at a low temperature. Several methods to improve the stability of bicelles, and to extend the ranges of alignment temperatures, using unsaturated/modified lipids or chemical additives have been reported28,29,30,31,32,33
. Amongst these NMR studies, the lowest temperature at which aligned bicelles used to study proteins is 25°C34
. In this study, we report the experimental conditions for the preparation of temperature resistant bicelles
and demonstrate their use in the structural studies of a full-length mammalian microsomal cytochrome P450 2B4.
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 blowtorch35,36,37,38,39,40,41,42
. 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. Microsomal cytochrome P450 2B4 has the molecular weight of 55.7 kDa and consists of a catalytic heme-containing soluble domain and a hydrophobic transmembrane domain. The transmembrane region of microsomal cytochromes P450 is essential for their functions because the lack of this membrane anchor results in a decrease to only 40% of all enzymatic activities. What the role of the transmembrane structure for enzymatic mechanisms is and how the membrane-associated region of the enzyme interacts with lipid bilayers are key questions to be addressed in order to explain how cytochrome P450s take hydrophobic compounds into their reaction centers. Despite its importance, the atomic-level transmembrane structure of microsomal cytochrome P450 has not been revealed experimentally, since its first identification in 196243
. Major difficulties in the structure determination of full-length microsomal cytochrome P450 include: (i) optimization of conditions to satisfy its thermal stability on both a larger soluble domain and a relatively smaller hydrophobic transmembrane domain simultaneously for crystallization, (ii) challenges related to the colossal molecular weight for solution NMR spectroscopy, and (iii) its thermal instability during biophysical experiments including solid-state NMR spectroscopy. In this study, we report the first study on the transmembrane structure and topology of the functional form of a heat sensitive cytochrome P450, using a newly developed temperature resistant bicelle compositions in a low temperature environment by means of solid-state NMR spectroscopy.