The native conformation of the human VDAC1 N terminus.

The voltage-dependent anion channel (VDAC) is located in the mitochondrial outer membrane and constitutes the major pathway for the transport of ADP, ATP, and other metabolites. It is also considered a key player in mitochondrial apoptosis. 2] Recently, the three-dimensional structure of the VDAC1 isoform was elucidated independently by three different experimental approaches. All structures reveal a novel 19-stranded b-barrel architecture with an N-terminal a helix positioned horizontally inside the pore. Although all three structures are highly similar in terms of the b barrel, they exhibit clear differences in the functionally important Nterminal region (Figure 1a). There is a general consensus that the N terminus is involved in the voltage-dependent gating process of the channel and that it may adopt different conformations depending on external factors. Based on these structural data, different models for voltage gating have been proposed. Ujwal et al. suggested that the whole helix may move towards the middle of the pore and thereby close the channel. On the other hand, as residues 11–20 are difficult to observe in solution-state NMR, Hiller and Wagner argued that they may be subject to conformational exchange and that movements in this part of the N terminus alone may explain the gating behavior. Alternatively, larger conformational rearrangements upon voltage gating that also involve the b barrel have been suggested based on electron microscopy and electrophysiological data. Additionally, some previous biophysical findings are at odds with all three published structures, and the question has been raised as to whether they really represent the native conformation in a natural membrane environment. In particular, N-terminal truncation mutants of VDAC1 were found to exhibit lower conductance than the full-length channel, which has been taken as an indication that the N-terminal helix may not lie inside the pore, but form part of the barrel wall. Solid-state NMR spectroscopy has proven to be a very useful method for structural investigations of membrane proteins in a natural lipid environment (see, for example, reference [13] for a recent review). We therefore investigated functional human VDAC1 in lipid bilayers using solid-state NMR spectroscopy, with a focus on the conformation of its N terminus. Functionality of our hVDAC1 preparation was confirmed by electrophysiological measurements in lipid bilayers (see the Supporting Information, Figure S1). Figure 1b shows a C–C proton-driven spin diffusion (PDSD) correlation spectrum of uniformly [C, N] isotope-labeled hVDAC1 reconstituted into dimyristoylphosphocholine (DMPC) liposomes. The spectra exhibit excellent sensitivity and resolution. A prediction of the Ca–Cb region of the spectrum based on the crystal structure of mouse VDAC1 (Supporting Information, Figure S2) agrees very well with the spectrum and indicates that the overall 19-stranded b-barrel fold is conserved in liposomes. Comprising 283 amino acids, VDAC1 is a challenging protein for solid-state NMR spectroscopy. To identify the N-terminal residues, we made use of different isotope labeling schemes, involving a (Lys, Trp, Tyr, Val) reverse-labeled and an (Ala, Asp, Leu, Val) forward-labeled protein variant along with the uniformly isotope-labeled sample. Furthermore, we studied an N-terminally truncated hVDAC1 variant (D(1-20)-hVDAC1). Figure 1c illustrates how a combination of results from the different samples was used in the assignment process. With a large set of homoand heteronuclear correlation spectra of the different protein variants (see, for example, the Supporting Information, Figure S3) we were able to obtain unambiguous de novo sequential resonance assignments for residues Ala2–Val17 (Supporting Information, Figure S4 and Table S1). All of these residues give rise to distinct narrow cross-peaks in experiments based on dipolar transfer schemes at sample temperatures between + 5 and + 25 8C (Supporting Information, Figure S5 and S6). This result demonstrates that the hVDAC1 N terminus assumes a well-defined conformation in liposomes and does not exhibit sizable dynamics on the sub-millisecond timescale. No peak broadening or doubling owing to chemical exchange on slower timescales was [*] Dr. R. Schneider, K. Giller, V. Daebel, Prof. Dr. M. Zweckstetter, Prof. Dr. C. Griesinger, Dr. S. Becker, Dr. A. Lange Department for NMR-based Structural Biology Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 G ttingen (Germany) Fax: (+ 49)551-201-2202 E-mail: adla@nmr.mpibpc. mpg.de Homepage: http://www.mpibpc. mpg.de/english/research/ags/ lange/