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Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

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Abstract

The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for 13C line widths and <0.5 ppm 15N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via 1H detected MAS NMR.

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Acknowledgments

This research was supported by NIH Grants EB001960 and EB002026.

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  1. Matthew T. Eddy

    Present address: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA

Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    Matthew T. Eddy, Yongchao Su, Robert Silvers, Loren Andreas, Lindsay Clark & Robert G. Griffin

  2. Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    Matthew T. Eddy, Yongchao Su, Robert Silvers, Loren Andreas, Lindsay Clark & Robert G. Griffin

  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Gerhard Wagner

  4. Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques (CNRS, ENS Lyon, UCB Lyon 1), Université de Lyon, 69100, Villeurbanne, France

    Loren Andreas, Guido Pintacuda & Lyndon Emsley

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Matthew T. Eddy and Yongchao Su have contributed equally to this article.

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Eddy, M.T., Su, Y., Silvers, R. et al. Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR. J Biomol NMR 61, 299–310 (2015). https://doi.org/10.1007/s10858-015-9903-1

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