Article

Solid-State Magic-Angle Spinning NMR of Outer-Membrane Protein G from Escherichia coli

Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
ChemBioChem (Impact Factor: 3.09). 09/2005; 6(9):1679-84. DOI: 10.1002/cbic.200500132
Source: PubMed

ABSTRACT

Uniformly 13C-,15N-labelled outer-membrane protein G (OmpG) from Escherichia coli was expressed for structural studies by solid-state magic-angle spinning (MAS) NMR. Inclusion bodies of the recombinant, labelled protein were purified under denaturing conditions and refolded in detergent. OmpG was reconstituted into lipid bilayers and several milligrams of two-dimensional crystals were obtained. Solid-state MAS NMR spectra showed signals with an apparent line width of 80-120 Hz (including homonuclear scalar couplings). Signal patterns for several amino acids, including threonines, prolines and serines were resolved and identified in 2D proton-driven spin-diffusion (PDSD) spectra.

Download full-text

Full-text

Available from: Ludwig Krabben
  • Source
    • "Higher concentrations have been achieved for example by preparing 3D crystals of the membrane protein DGK [24] or by precipitating the transmembrane enzyme DsbB [25]. However, the best way to ensure a membrane environment at high protein concentration is 2D crystallisation, as demonstrated for the β-barrel outer-membrane porin OmpG [26]. Our crystallisation screen has shown that it is possible to crystallize PR under wide variety of a few hundred conditions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Proteins of the proteorhodopsin (PR) family are found abundantly in many marine bacteria in the photic zone of the oceans. They are colour-tuned to their environment. The green absorbing species has been shown to act as a light-driven proton pump and thus could form a potential source of energy. The pK(a) of the primary proton acceptor is close to the pH of seawater which could also indicate a regulatory role. Here, we review and summarize our own recent findings in the context of known data and present some new results. Proton transfer in vitro by PR is shown by a fluorescence assay which confirms a pH dependent vectoriality. Previously reported low diffracting 2D crystal preparations of PR are assessed for their use for solid-state NMR by two dimensional (13)C-(13)C DARR spectra. (15)N-(1)H HETCOR MAS NMR experiments show bound water in the vicinity of the protonated Schiff base which could play a role in proton transfer. The effect of highly conserved H75 onto the properties of the chromophore has been investigated by single site mutations. They do show a pronounced effect onto the optical absorption maximum and the pK(a) of the proton acceptor but have only a small effect onto the (15)N chemical shifts of the protonated Schiff base.
    Full-text · Article · Apr 2009 · Biochimica et Biophysica Acta
  • Source
    • "Methodological and conceptual improvements, however, are still necessary for a straight forward and routine application of solid-state NMR to biological matter. Nevertheless, its applicability to a wide range of candidates for biomolecular investigation like uniformly isotopically enriched membrane proteins [3] [4] [5] [6] or proteins forming aggregates [7] [8] [9] [10] [11] [12] has been demonstrated. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Assignment of proteins in MAS (magic angle spinning) solid-state NMR relies so far on correlations among heteronuclei. This strategy is based on well dispersed resonances in the (15)N dimension. In many complex cases like membrane proteins or amyloid fibrils, an additional frequency dimension is desirable in order to spread the amide resonances. We show here that proton detected HNCO, HNCA, and HNCACB type experiments can successfully be implemented in the solid-state. Coherences are sufficiently long lived to allow pulse schemes of a duration greater than 70 ms before incrementation of the first indirect dimension. The achieved resolution is comparable to the resolution obtained in solution-state NMR experiments. We demonstrate the experiments using a triply labeled sample of the SH3 domain of chicken alpha-spectrin, which was re-crystallized in H(2)O/D(2)O using a ratio of 1/9. We employ paramagnetic relaxation enhancement (PRE) using EDTA chelated Cu(II) to enable rapid data acquisition.
    Preview · Article · Aug 2008 · Journal of Magnetic Resonance
  • Source
    • "Higher concentrations have been achieved for example by preparing 3D crystals of the membrane protein DGK [18] or by precipitating the transmembrane enzyme DsbB [19]. However, the best way to ensure a membrane environment at high protein concentration is 2D crystallisation, as demonstrated for the β-barrel outer-membrane porin OmpG [20]. We have therefore prepared isotope labelled green proteorhodopsin as 2D crystals and report a first characterisation of these samples by electron microscopy and solid state NMR. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Proteorhodopsin (PR) a recent addition to retinal type 1 protein family, is a bacterial homologue of archaeal bacteriorhodopsin. It was found to high abundance in gamma-proteobacteria in the photic zone of the oceans and has been shown to act as a photoactive proton pump. It is therefore involved in the utilisation of light energy for energy production within the cell. Based on data from biodiversity screens, hundreds of variants were discovered worldwide, which are spectrally tuned to the available light at different locations in the sea. Here, we present a characterisation of 2D crystals of the green variant of proteorhodopsin by electron microscopy and solid state NMR. 2D crystal formation with hexagonal protein packing was observed under a very wide range of conditions indicating that PR might be also closely packed under native conditions. A low-resolution 2D projection map reveals a ring-shaped oligomeric assembly of PR. The protein state was analysed by 15N MAS NMR on lysine, tryptophan and methionine labelled samples. The chemical shift of the protonated Schiff base was almost identical to non-crystalline preparations. All residues could be cross-polarised in non-frozen samples. Lee-Goldberg cross-polarisation has been used to probe protein backbone mobility.
    Full-text · Article · Jan 2008 · Biochimica et Biophysica Acta
Show more