Article

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
Source: PubMed
6 Followers
 · 
113 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396-10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure-activity correlation experiments across a wide range of timescales.
    Journal of Biomolecular NMR 01/2015; DOI:10.1007/s10858-014-9893-4 · 3.31 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: For many membrane proteins the determination of their topology remains a challenge for methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Electron paramagnetic resonance (EPR) spectroscopy has evolved as an alternative technique to study structure and dynamics of membrane proteins. The present study demonstrates the feasibility of membrane protein topology determination using limited EPR distance and accessibility measurements. The BCL::MP-Fold (BioChemical Library membrane protein fold) algorithm assembles secondary structure elements (SSEs) in the membrane using a Monte Carlo Metropolis (MCM) approach. Sampled models are evaluated using knowledge-based potential functions and agreement with the EPR data and a knowledge-based energy function. Twenty-nine membrane proteins of up to 696 residues are used to test the algorithm. The RMSD100 value of the most accurate model is better than 8Å for twenty-seven, better than 6Å for twenty-two and better than 4Å for fifteen out of twenty-nine proteins, demonstrating the algorithms ability to sample the native topology. The average enrichment could be improved from 1.3 to 2.5, showing the improved discrimination power by using EPR data.
    Proteins Structure Function and Bioinformatics 03/2015; DOI:10.1002/prot.24801 · 2.92 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: CLC transporters catalyze the exchange of Cl(-) for H(+) across cellular membranes. To do so, they must couple Cl(-) and H(+) binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state (13)C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H(+)) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H(+)-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl(-)-permeation pathway, to the extracellular solution. The H(+)-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H(+) binding is mechanistically coupled to closing of the intracellular access-pathway for Cl(-).
    Journal of Biomolecular NMR 01/2015; 61(3-4). DOI:10.1007/s10858-015-9898-7 · 3.31 Impact Factor

Full-text (2 Sources)

Download
22 Downloads
Available from
Dec 4, 2014