Nonmicellar systems for solution NMR spectroscopy of membrane proteins

Harvard Medical School, Boston, MA 02115, USA.
Current Opinion in Structural Biology (Impact Factor: 7.2). 08/2010; 20(4):471-9. DOI: 10.1016/
Source: PubMed


Integral membrane proteins play essential roles in many biological processes, such as energy transduction, transport of molecules, and signaling. The correct function of membrane proteins is likely to depend strongly on the chemical and physical properties of the membrane. However, membrane proteins are not accessible to many biophysical methods in their native cellular membrane. A major limitation for their functional and structural characterization is thus the requirement for an artificial environment that mimics the native membrane to preserve the integrity and stability of the membrane protein. Most commonly employed are detergent micelles, which can however be detrimental to membrane protein activity and stability. Here, we review recent developments for alternative, nonmicellar solubilization techniques, with a particular focus on their application to solution NMR studies. We discuss the use of amphipols and lipid bilayer systems, such as bicelles and nanolipoprotein particles (NLPs). The latter show great promise for structural studies in near native membranes.

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Available from: Sebastian Hiller
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    • "A potential solution is offered by the use of isotropic bicelles or nanodiscs, which are larger than micelles, but still soluble. All of these three systems have been used successfully for liquid-state NMR (Raschle et al, 2010), but proteins of the size of full length ABC transporters pose a general problem due to their molecular weight. "
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    ABSTRACT: ABC transporters form a superfamily of integral membrane proteins involved in translocation of substrates across the membrane driven by ATP hydrolysis. Despite available crystal structures and extensive biochemical data, many open questions regarding their transport mechanisms remain. Therefore, there is a need to explore spectroscopic techniques such as solid state NMR in order to bridge the gap between structural and mechanistic data. In this study, we investigate the feasibility of using E. coli MsbA as a model ABC transporter for solid state NMR studies. We show that optimised solubilisation and reconstitution procedures enable preparing stable and homogenous protein samples. Depending on the solubilisation times, MsbA can be obtained in either an apo- or in a native lipid-A bound form. Building onto these optimizations, the first promising MAS-NMR spectra with narrow lines have been recorded. However, further sensitivity improvements are required so that complex NMR experiments can be recorded within a reasonable amount of time. We therefore demonstrate the usability of paramagnetic doping for rapid data acquisition and explore dynamic nuclear polarisation as a method for general signal enhancement. Our results demonstrate that solid state NMR provides an opportunity to address important biological questions related to complex mechanisms of ABC transporters.
    Full-text · Article · Apr 2015 · Biological Chemistry
    • "Elter et al. 2014; Planchard et al. 2014). As of today, the approach has been applied to five MPs, three b-barrel ones, OmpX (Bazzacco et al. 2012; Catoire et al. 2010b, 2009; Etzkorn et al. 2014) and the transmembrane domains of OmpA from E. coli (Dahmane et al. 2011; Zoonens et al. 2005) and from Klebsiella pneumoniae (Planchard et al. 2014; Renault 2008), and two a-helical ones, BR (Etzkorn et al. 2013, 2014; Raschle et al. 2010) and the LTB2 leukotriene receptor (Catoire et al. 2011, 2010a).Whereas MP/APol complexes are slightly bigger objects than the smallest MP/ detergent ones, and therefore solution NMR spectra at a given temperature can be slightly less well resolved (Planchard et al. 2014; Zoonens et al. 2005), this is more than compensated for by the higher stability of APoltrapped MPs, as well as by the relative ease with which A8–35 can be partially (Gohon et al. 2004) or totally (Giusti et al. 2014) deuterated, giving access, for instance, to the determination of 1 H– 1 H distances by nuclear Overhauser effect measurements (Catoire et al. 2010b; Planchard et al. 2014). "
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    ABSTRACT: Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3-14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3-14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of (15)N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3-14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations.
    No preview · Article · Jun 2014 · Journal of Membrane Biology
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    • "As of now, no MP structure has been solved de novo by NMR using APols as the solubilizing medium. That is indeed possible is shown by (1) the high resolution of TROSY 1 H, 15 N 2D spectra (Raschle et al. 2010; Etzkorn et al. 2013; Dahmane et al. 2011; Bazzacco et al. 2012; Zoonens et al. 2005; Catoire et al. 2010a) (Fig. 2) and (2) the fact that high-resolution 3D experiments can be carried out within a reasonable time span (Etzkorn et al 2014) (Fig. 3). All APols tested to date for use in solution NMR [A8-35 (Zoonens et al. 2005), SAPols (Dahmane et al. 2011), NAPols (Bazzacco et al. 2012)] form with MPs complexes of a similar size and can be used for backbone assignments. "
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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.
    Full-text · Article · Mar 2014 · Journal of Membrane Biology
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