High-level expression, refolding and probing the natural fold of the human voltage-dependent anion channel isoforms I and II.
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.
Engelhardt
Harald
H
Department of Molecular Structure Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152, Martinsried, Germany.
Meins
Thomas
T
Poynor
Melissa
M
Adams
Volker
V
Nussberger
Stephan
S
Welte
Wolfram
W
Zeth
Kornelius
K
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry* / Q000235:genetics; D050996:Voltage-Dependent Anion Channel 2 / Q000737:chemistry* / Q000235:genetics