Recent Advances in the Application of Solution NMR Spectroscopy to Multi-Span Integral Membrane Proteins.

Korea Polar Research Institute, Korea Ocean Research and Development Institute, Incheon, 406-840, Korea.
Progress in Nuclear Magnetic Resonance Spectroscopy (Impact Factor: 8.71). 11/2009; 55(4):335-360. DOI: 10.1016/j.pnmrs.2009.07.002
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    ABSTRACT: Spontaneously forming structures of a system composed of dimyristoyl phosphatidylcholine (DMPC) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) were studied by small angle neutron scattering (SANS), (31)P NMR and stimulated echo (STE) pulsed field gradient (PFG) (1)H NMR diffusion measurements. The charged lipid dimyristoyl phosphatidylglycerol (DMPG) was used to induce different surface charge densities. The structures adopted were investigated as a function of temperature and lipid concentration for samples with a constant molar ratio of long-chain to short-chain lipid (=3). In the absence of DMPG, zwitterionic bicellar mixtures exhibited a phase transition from discoidal bicelles, or ribbons, to multi-lamellar vesicles, either upon dilution or with increased temperature. CHAPSO-containing mixtures showed a higher thermal stability in morphology than DHPC-containing mixtures at the corresponding lipid concentrations. In the presence of DMPG, discoidal bicelles (or ribbons) were also found at low temperature and lower lipid concentration mixtures. At high temperature perforated lamellae were observed in high concentration mixtures (≥ 7.5 wt%), while uniform unilamellar vesicles and bicelles formed in low-concentration mixtures (≤ 2.5 wt%), respectively, when the mixtures were moderately and highly charged. From the results, spontaneous structural diagrams of the zwitterionic and charged systems were constructed.
    Langmuir 09/2013; · 4.38 Impact Factor
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    ABSTRACT: Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [15N-1H]- and [13C-1H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan 15N backbone positions and also resolved signals for 15N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [13C-1H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.
    Molecular Membrane Biology 04/2014; 31(4):131-140. · 1.73 Impact Factor
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    ABSTRACT: Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) mediating fast neurotransmission in the central and peripheral nervous systems. They are important targets for many currently used clinical drugs, such as general anesthetics, and for allosteric modulators with potential therapeutic applications. Here, we provide an overview of advances in the use of solution NMR in structural and dynamic characterization of ion channels, particularly human Cys-loop receptors. We present challenges to overcome and realistic solutions for achieving high-resolution structural information for this family of receptors. We discuss how subtle structural differences among homologous channels define unique channel pharmacological properties and advocate the necessity to determine high-resolution structures for individual receptor subtypes. Finally, we describe drug binding to the Cys-loop receptors' TMD identified by solution NMR and the associated dynamics changes relevant to channel functions. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces.
    Biochimica et Biophysica Acta 03/2014; · 4.66 Impact Factor

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