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The voltage-dependent anion channel (VDAC) is the major protein found in the outer membrane of mitochondria. The channel is responsible for the exchange of ATP/ADP and the translocation of ions and other small metabolites over the membrane. In order to obtain large amounts of pure and suitably folded human VDAC for functional and structural studies, the genes of the human isoforms I and II (HVDAC1 and HVDAC2) were cloned in Escherichia coli. High-level expression led to inclusion body formation. Both proteins could be refolded in vitro by adding denatured protein to a solution of zwitterionic or nonionic detergents. A highly efficient and fast protocol for refolding was developed that yielded more than 50 mg of pure human VDACs per liter of cell culture. The native and functional state of the refolded porins was probed by Fourier transform infrared spectroscopy to determine the secondary structure composition and by electrophysiological measurements, demonstrating the pore-forming activity of HVDAC1. Furthermore, binding of HVDAC1 to immobilized ATP was demonstrated. Limited proteolysis of HVDAC1 protein embedded in detergent micelles in combination with matrix-assisted laser desorption ionization mass spectrometric analysis was applied to identify micelle-exposed regions of the protein and to develop an improved topology model. Our analysis strongly suggests a 16-stranded, antiparallel beta-barrel with one large and seven short loops and turns. Initial crystallization trials of the protein yielded crystals diffracting to 8 Angstrom resolution.

The protein translocase of the outer mitochondrial membrane (TOM) serves as the main entry site for virtually all mitochondrial proteins. Like many other protein translocases it also has an ion channel activity that can be used to study the dynamical properties of this supramolecular complex. We have purified TOM core complex and Tom40, the main pore forming subunit, from mitochondria of the filamentous fungus Neurospora crassa and incorporated them into planar lipid bilayers. We then examined their single channel properties to provide a detailed description of the conformational dynamics of this channel in the absence of its protein substrate. For isolated TOM core complex we have found at least six conductance states. Transitions between these states were voltage-dependent with a bell-shaped open probability distribution and distinct kinetics depending on the polarity of the applied voltage. The states with the largest conductance followed an Ohmic I/V characteristic consistent with a large cylindrical pore with very little interaction with the permeating ions. For the lower conductance states, however, we have observed inverted S-shaped nonlinear current-voltage curves reminiscent to those of much narrower pores where the permeating ions have to surmount an electrostatic energy barrier. At low voltages (<+/-70 mV), purified Tom40 protein did not show any transitions between its conductance states. Prolonged exposure to higher voltages induced similar gating behavior to what we observed for TOM core complex. This effect was time-dependent and reversible, indicating that Tom40 forms not only the pore but also contains the "gating machinery" of the complex. However, for proper functioning, additional proteins (Tom22, Tom7, Tom6, and Tom5) are required that act as a modulator of the pore dynamics by significantly reducing the energy barrier between different conformational states.

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