Optimized Phospholipid Bilayer Nanodiscs Facilitate High-Resolution Structure Determination of Membrane Proteins
ABSTRACT Structural studies of membrane proteins are still hampered by difficulties of finding appropriate membrane mimicking media that maintain protein structure and function. Phospholipid nanodiscs seem promising to overcome the intrinsic problems of detergent containing environments. While nanodiscs can offer a near native environment, the large particle size complicates their routine use in the structural analysis of membrane proteins by solution NMR. Here, we introduce nanodiscs assembled from shorter ApoA-I protein variants that are of markedly smaller diameter and show that the resulting discs provide critical improvements for the structure determination of membrane proteins by NMR. Using the bacterial outer membrane protein OmpX as an example, we demonstrate that the combination of small nanodisc size, high deuteration levels of protein and lipids and the use of advanced non-uniform NMR sampling methods enable the NMR resonance assignment as well as the high-resolution structure determination of polytopic membrane proteins in a detergent-free, near-native lipid bilayer setting. By applying this method to bacteriorhodopsin we show that our smaller nanodiscs can also be beneficial for the structural characteri-zation of the important class of seven-transmembrane helical proteins. Our set of engineered nanodiscs of subsequently smaller diameters can be used to screen for optimal NMR spectral quality for any given membrane protein while still providing a functional environment. In addi-tion to their key improvements for de novo structure determination, due to their smaller size these nanodiscs enable the investigation of inter-actions between membrane proteins and their (soluble) partner proteins, unbiased by the presence of detergents that might disrupt biological relevant interactions.
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ABSTRACT: Solution-state nuclear magnetic resonance studies of membrane proteins are facilitated by the increased stability that trapping with amphipols confers to most of them as compared to detergent solutions. They have yielded information on the state of folding of the proteins, their areas of contact with the polymer, their dynamics, water accessibility, and the structure of protein-bound ligands. They benefit from the diversification of amphipol chemical structures and the availability of deuterated amphipols. The advantages and constraints of working with amphipols are discussed and compared to those associated with other non-conventional environments, such as bicelles and nanodiscs.Journal of Membrane Biology 03/2014; 247(9-10). DOI:10.1007/s00232-014-9654-z · 2.17 Impact Factor
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ABSTRACT: NMR spectroscopy of helical membrane proteins has been very challenging on multiple fronts. The expression and purification of these proteins while maintaining functionality has consumed countless graduate student hours. Sample preparations have depended on whether solution or solid-state NMR spectroscopy was to be performed – neither have been easy. In recent years it has become increasingly apparent that membrane mimic environments influence the structural result. Indeed, in these recent years we have rediscovered that Nobel laureate, Christian Anfinsen, did not say that protein structure was exclusively dictated by the amino acid sequence, but rather by the sequence in a given environment (C.B. Anfinsen, 1973, Science 181:223-230). The environment matters, molecular interactions with the membrane environment are significant and many examples of distorted, non-native membrane protein structures have recently been documented in the literature. However, solid-state NMR structures of helical membrane proteins in proteoliposomes and bilayers are proving to be native structures that permit a high resolution characterization of their functional states. Indeed, solid-state NMR is uniquely able to characterize helical membrane protein structures in lipid environments without detergents. Recent progress in expression, purification, reconstitution, sample preparation and in the solid-state NMR spectroscopy of both oriented samples and magic angle spinning samples has demonstrated that helical membrane protein structures can be achieved in a timely fashion. Indeed, this is a spectacular opportunity for the NMR community to have a major impact on biomedical research through the solid-state NMR spectroscopy of these proteins.Journal of Magnetic Resonance 01/2013; 239. DOI:10.1016/j.jmr.2013.12.006 · 2.32 Impact Factor
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ABSTRACT: Cannabinoid receptor CB2 is a seven transmembrane-domain integral membrane protein that belongs to a large superfamily of G protein-coupled receptors (GPCR). CB2 is a part of the endocannabinoid system that plays vital role in regulation of immune response, inflammation, pain sensitivity, obesity and other physiological responses. Information about the structure and mechanisms of functioning of this receptor in cell membranes is essential for the rational development of specific pharmaceuticals. Here we review the methodology for recombinant expression, purification, stabilization and biochemical characterization of CB2 suitable for preparation of multi-milligram quantities of functionally active receptor. The biotechnological protocols include expression of the recombinant CB2 in E. coli cells as a fusion with the maltose binding protein, stabilization with a high affinity ligand and a derivative of cholesterol in detergent micelles, efficient purification by tandem affinity chromatography, and reconstitution of the receptor into lipid bilayers. The purified recombinant CB2 receptor is amenable to functional and structural studies including nuclear magnetic resonance spectroscopy and a wide range of biochemical and biophysical techniques.03/2013; 6(7):e201303011. DOI:10.5936/csbj.201303011