Structure and Orientation of Two Voltage-dependent Anion-selective Channel Isoforms

Abstract

Two VDAC (voltage-dependent anion-selective channel) isoforms were purified from seed cotyledons of Phaseolus vulgaris by chromatofocusing chromatography. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to study the structural properties of the two isoforms reconstituted in a mixture of asolectin and 5% stigmasterol. The IR spectra of the two VDAC isoforms were highly similar indicating 50 to 53% anti-parallel ␤-sheet. The orientation of the ␤-strands relative to the barrel axis was calculated from the experimentally obtained dichroic ratios of the amide I ␤-sheet component and the amide II band. Comparing the IR spectra of the reconstituted VDAC isoforms with the IR spectra of the bacterial porin OmpF, for which a high resolution structure is available, provided evidence for a general structural organization of the VDAC isoforms similar to that of bacterial porins. Hydrogen-deuterium exchange measurements indicated that the exchange of the amide protons occurs to a higher extent in the two VDAC isoforms than in the OmpF porin.Mitochondria are surrounded by two distinct membranes. The mitochondrial outer membrane is freely permeable for small hydrophilic solutes up to approximately 6 kDa. This property has been attributed to the presence of a pore forming protein, the voltage-dependent anion-selective channel (VDAC) 1 or mitochondrial porin (1). Biophysical properties of VDAC channels inserted in planar lipid bilayers have been studied in a large variety of organisms. VDAC channels switch from a high conducting fully open state (3.6 to 4.5 nS in 1 M KCl) at low voltages to several low-conducting substates upon application of voltages higher than Ϯ20 mV. VDAC is slightly anion selective in the fully open state but switches to cation selectivity in the low-conducting substates (2). In these substates, the flow of negatively charged metabolites such as succinate, citrate, phosphate (3), and adenine nucleotides (4, 5) through the channel is strongly reduced compared with the fully open state. Therefore, VDAC is thought to regulate the mitochondrial metabolism by regulating the flux of metabolites across the mitochondrial outer membrane.VDAC channels are involved in different cellular events like the induced release of cytochrome c, which constitutes an early step in apoptosis (6). VDAC is the binding site for cytoplasmic enzymes like glycerol kinase and hexokinase (7) and for the cytoskeleton (8). The different VDAC isoforms that exist in yeast, plants, and mammals could be involved in specific functions. Two human VDAC isoforms have different binding affinities for hexokinase (9). Plant VDAC isoforms have slightly different electrophysiological properties (10), and mitochondria isolated from yeast vdac minus mutants show differences in their outer membrane permeability to NADH depending on which of the three mouse VDAC isoforms was expressed (11). Therefore, it seems reasonable to assume that the multitude of functional properties that are attributed to VDAC could arise from ...

Voltage-dependent structural changes of the membrane-bound anion channel hVDAC1 probed by SEIRA and electrochemical impedance spectroscopy

“…At first glance, this amide II band pattern seems to be in contradiction to the previous ATR-IR spectra of hVDAC1 that show a broad amide II band centred at 1530 cm À1 . 21 In the case of SEIRA spectroscopy, however, the preferential enhancement for structural elements in close proximity to the Au surface has to be taken into account. These are mainly b-sheet-connecting turns and random coils.…”

Determination of the Conformation of the Human VDAC1 N‐Terminal Peptide, a Protein Moiety Essential for the Functional Properties of the Pore

“…[34,60] The N-terminal sequence is one of the most immunogenic parts of the VDAC. [26,34,55] Monoclonal antibodies against this peptide have been produced, and these antibodies invariably detected this moiety outside the membrane.…”

Correct Folding of the β-Barrel of the Human Membrane Protein VDAC Requires a Lipid Bilayer

“…6,7 Both, the exact number of strands in the β-barrel domains of the various VDACs and the arrangement of the helix are a matter of debate. Alternatively, a 16-stranded β-barrel has been proposed 10,11 with the Nterminal amphipathic helix located predominantly at the membrane water-interface or outside the membrane. The topology may also depend on the species.…”

“…Circular dichroism (CD) and infrared spectroscopic studies indicated that VDACs of various origins contain between 9 and 27% α-helical and between 30 and 53% β-sheet structure. 10,12,13 Bacterial porins of similar size like OmpF and FomA, contain only ∼ 2 to 5% helical structure according to X-ray crystallography (2OMF) 14 or CD spectroscopy. 15 The majority of studies on the structure of VDAC have been performed in detergent micelles, while VDAC function was assayed in lipid bilayers.…”

“…Analysis II was performed on hVDAC1 that was first solubilized in LDAO and then reconstituted into diC 12:0 PC (the CD spectrum is shown in Figure 1 3,5,7,9,12,14,16), and in diC 12:0 PC (lanes 4,6,8,10,13,15,17), indicate formation of a single fragment of ∼ 26 to 27 kDa size in diC 12:0 PC bilayers that is present after 240 min of proteolysis. In LDAO, hVDAC1 is first cleaved into two major fragments of ∼ 26 to 27 kDa and of ∼ 24 kDa.…”