Optimized Phospholipid Bilayer Nanodiscs Facilitate High-Resolution Structure Determination of Membrane Proteins

Journal of the American Chemical Society (Impact Factor: 12.11). 01/2013; 135(5). DOI: 10.1021/ja310901f
Source: PubMed


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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    • "For the purposes of antibody phage display, nanodiscs allow the protein targets to be reconstituted into a native-like lipid environment with access to the protein from both sides of the membrane, and enable the user to bypass difficulties in sample handling, thus increasing the overall stability of the antigen (Bayburt et al., 2006). The diameter and chemical makeup of nanodiscs can be adjusted during protein reconstitution by using different variants of MSP and various lipid compositions (Ritchie et al., 2009; Hagn et al., 2013). Importantly, any modifications of the antigens necessary for the phage display experiment, such as biotinylation, can be readily achieved by attachment through MSP or lipid modifications, leaving the membrane protein unaltered. "
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    ABSTRACT: A major challenge in membrane biophysics is to define the mechanistic linkages between a protein's conformational transitions and its function. We describe a novel approach to stabilize transient functional states of membrane proteins in native-like lipid environments allowing for their structural and biochemical characterization. This is accomplished by combining the power of antibody Fab-based phage display selection with the benefits of embedding membrane protein targets in lipid-filled nanodiscs. In addition to providing a stabilizing lipid environment, nanodiscs afford significant technical advantages over detergent-based formats. This enables the production of a rich pool of high-performance Fab binders that can be used as crystallization chaperones, as fiducial markers for single-particle cryoelectron microscopy, and as probes of different conformational states. Moreover, nanodisc-generated Fabs can be used to identify detergents that best mimic native membrane environments for use in biophysical studies.
    Full-text · Article · Dec 2015 · Structure
    • "Reconstitution of MPs into these soluble particles seems to be generically applicable irrespective of the type of protein and they convey a relatively high protein stability (for reviews see Bayburt and Sligar 2010; Schuler et al. 2012). The diameter of nanodiscs is typically in the order of ~10 nm, but generation of specific MSP variants allows the formation of smaller (~6–7 nm) (Hagn et al. 2013; Wang et al. 2015) and larger (16–17 nm) (Grinkova et al. 2010) nanodiscs. Furthermore , the use of different apolipoproteins or derived peptides and the variation of the peptide/protein–lipid ratio enable the preparation of larger particles (Chromy et al. 2007; Park et al. 2011). "
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    ABSTRACT: A new and promising tool in membrane research is the detergent-free solubilization of membrane proteins by styrene-maleic acid copolymers (SMAs). These amphipathic molecules are able to solubilize lipid bilayers in the form of nanodiscs that are bounded by the polymer. Thus, membrane proteins can be directly extracted from cells in a water-soluble form while conserving a patch of native membrane around them. In this review article, we briefly discuss current methods of membrane protein solubilization and stabilization. We then zoom in on SMAs, describe their physico-chemical properties, and discuss their membrane-solubilizing effect. This is followed by an overview of studies in which SMA has been used to isolate and investigate membrane proteins. Finally, potential future applications of the methodology are discussed for structural and functional studies on membrane proteins in a near-native environment and for characterizing protein-lipid and protein-protein interactions.
    No preview · Article · Dec 2015 · European Biophysics Journal
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    • "Several manuscripts explore the use on phospholipid nanodiscs for studies of integral membrane proteins. Wagner's group has recently introduced small phospholipid nanodiscs to obtain well-resolved liquid state NMR spectra of membrane proteins (Hagn et al. 2013; Raschle et al. 2009). Here they use these nanodiscs to determine a high-resolution structure of the b-barrel protein OmpX (Hagn and Wagner 2014). "
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    ABSTRACT: The functional importance of proteins that interact with biological membranes can hardly be overestimated. About half of the medicinal drug targets are membrane proteins. Nevertheless, the structural biology of these proteins has been very challenging as only a little more than 500 unique membrane protein structures are present in the PDB (out of 100,000 structures) and also accessible in a membrane protein database ( About 16 % of these have been derived by NMR spectroscopy. While NMR is limited by molecular size it has the unparalleled capability of observing internal mobility, which can be analyzed at atomic resolution once resonance assignments are obtained. The main obstacles for assignment, dynamics studies and structure determination are low overexpression levels and a high content of hydrophobic amino acids, necessary for embedding into a biological membrane. Thus, some membrane mimetic must always be part of the protein preparation, both ...
    Preview · Article · Apr 2015 · Journal of Biomolecular NMR
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