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Science 321 (5893): 1206-1210

Copyright © 2008 by the American Association for the Advancement of Science

Solution Structure of the Integral Human Membrane Protein VDAC-1 in Detergent Micelles

Sebastian Hiller1, Robert G. Garces1*, Thomas J. Malia1*{dagger}, Vladislav Y. Orekhov1,3, Marco Colombini2, and Gerhard Wagner1{ddagger}

1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
2 Department of Biology, University of Maryland, College Park, MD 20742, USA.
3 Swedish NMR Centre, University of Gothenburg, Box 465, 40530 Gothenburg, Sweden.


Figure 1 Fig. 1.. Architecture of VDAC-1. (A) The amino acid sequence of VDAC-1 in one-letter code (36) is arranged according to the secondary and tertiary structure. Amino acids in squares denote β sheet secondary structure as identified by secondary chemical shifts; all other amino acids are in circles. Red and blue lines denote experimentally observed NOE contacts between two amide protons and NOE contacts involving side chain atoms, respectively. Bold lines indicate strong NOEs typically observed between hydrogen-bonded residues in β sheets. For clarity of the presentation, not all observed NOEs are shown. The 19th strand is duplicated at the right, next to strand 1, to allow for indicating the barrel-closure NOEs. The side chains of white and orange residues point toward the inside and the outside of the barrel, respectively. Dashed lines show probable contacts between protons with degenerate 1H chemical shifts. Gray residues could not be assigned so far. Every 10th amino acid is marked with a heavy outline, and corresponding residue numbers are indicated. (B) Strips from a 3D [1H,1H]-NOESY-15N-TROSY defining the barrel closure between parallel strands 1 and 19. Red lines show the interstrand contacts for the depicted residues, whereas the violet lines indicate the NOE contacts for the respective opposite residues. ppm, parts per million. (C) Strip from a3D [1H,1H]-NOESY-13C-HMQC (heteronuclear multiple-quantum coherence) taken at the position of a methyl group of Leu10. The assignments of the individual NOE signals are indicated on the left and exemplify the NOEs defining the location of the N-terminal helix in the barrel. The frequency axes {omega}1, {omega}2, and {omega}3 are indicated. [View Larger Version of this Image (66K GIF file)]
 

Figure 2 Fig. 2.. NMR solution structure of VDAC-1 in LDAO micelles. (A and B) Top and side views, respectively, of the conformer closest to the mean of the conformational ensemble in ribbon representation. β sheets are shown blue, and {alpha} helical secondary structures in red and yellow. N- and C-termini and residues L150 and V143 are indicated. (C) Van der Waals surface of VDAC-1. The surface is colored according to the surface potential, calculated by using vacuum electrostatics in the program PyMOL (38). Blue indicates positive charge, and red, negative charge. [View Larger Version of this Image (68K GIF file)]
 

Figure 3 Fig. 3.. Hydrophobic surface of VDAC-1. (A) Result of a titration with the spin-labeled detergent 16-DSA. Residues with a relaxation enhancement {epsilon} > 20 s–1 mM–1 are green (30). These residues are in close contact to the hydrophobic interior of the micelle. Residues with {epsilon} ≤ 20 s–1 mM–1 are white. Gray residues are unassigned. Residues 1 to 21 have been omitted; no interaction with the spin label was observed for these. (B and C) Surface plot of outer and inner surfaces of VDAC-1, respectively, with the side chains of the hydrophobic residues Leu, Val, Ile, Met, Phe, and Trp shown in yellow and all other residues in white. [View Larger Version of this Image (95K GIF file)]
 

Figure 4 Fig. 4.. Interactions of VDAC-1. In all three panels, the loop connecting strands 18 and 19 is indicated for orientation. (A) Residues with substantial chemical shift changes [{Delta}{delta}(HN) > 0.05 ppm] caused by cholesterol binding are shown in yellow (fig. S12). The amino acids of VDAC-1 are shown as in Fig. 1A. (B) Amide resonances of VDAC-1 with substantial chemical shift changes (fig. S13) caused by β-NADH are labeled magenta in this ribbon representation; all other residues are gray. (C) Residues involved in Bcl-xL binding (13) are marked red in this ribbon representation; all other residues are gray. [View Larger Version of this Image (83K GIF file)]
 


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