Magic Angle Spinning NMR Investigations of the Human Voltage Dependent Anion Channel

Abstract

In humans, transport of metabolites through the outer mitochondrial membrane is controlled by a 283 residue, 31 kDa pore-forming protein, the voltage-dependent anion-selective channel (VDAC). In addition to providing a main pathway for metabolite trafficking through the outer membrane, VDAC is postulated to play a critical role in cell apoptosis. VDAC interacts with the Bcl-2 family of pro-apoptotic and anti-apoptotic proteins that control the permeability of the outer membrane to apoptotic signals including the release of cytochrome c.Recently, the solution NMR structure of human VDAC-1 (hVDAC1) reconstituted in detergent micelles has been reported by two groups, revealing a 19-stranded β-barrel with a short α helix at the N terminus. While these structures are consistent with previous sequence analysis and biophysical studies, there are indications that interactions of the protein with a lipid bilayer are essential for its proper structure as well as function.Accordingly, we have undertaken studies on hVDAC1 reconstituted in DMPC bilayers with magic angle spinning (MAS) solid state NMR (SSNMR). hVDAC1 forms structurally homogeneous two-dimensional microcrystals as judged by high resolution 13C/15N MAS spectra. In addition, uniformly 13C and 15N labeled hVDAC1 in 2D DMPC cyrstals exhibit well resolved multidimensional MAS spectra that allow group assignments, structural analysis, and comparison of chemical shifts with the solution spectra.