title: Conservation of the oligomeric state of native VDAC1 in detergent micelles.
abstract: The voltage-dependent anion-selective channel (VDAC) is an intrinsic β-barrel membrane protein located within the mitochondrial outer membrane where it serves as a pore, connecting the mitochondria to the cytosol. The high-resolution structures of both the human and murine VDACs have been resolved by X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) in 2008. However, the structural data are not completely in line with the findings that were obtained after decades of research on biochemical and functional analysis of VDAC. This discrepancy may be related to the fact that structural biology studies of membrane proteins reveal specific static conformations that may not necessarily represent the physiological state. For example, overexpression of membrane proteins in bacterial inclusion bodies or simply the extraction from the native lipid environment using harsh purification methods (i.e. chaotropic agents) can disturb the physiological conformations and the supramolecular assemblies. To address these potential issues, we have developed a method, allowing rapid one step purification of endogenous VDAC expressed in the native mitochondrial membrane without overexpression of recombinant protein or usage of harsh chaotropic extraction procedures. Using the Saccharomyces cerevisiae isoform 1 of VDAC as a model, this method yields efficient purification, preserving VDAC in a more physiological, native state following extraction from mitochondria. Single particle analysis using transmission electron microscopy (TEM) demonstrated conservation of oligomeric assembly after purification. Maintenance of the native state was evaluated using functional assessment that involves an ATP-binding assay by micro-scale thermophoresis (MST). Using this approach, we were able to determine for the first time the apparent KD for ATP of 1.2 mM.
authors.affiliation: Institute of Biochemistry and Molecular Medicine (IBMM), National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland. Electronic address: benjamin.clemencon@ibmm.unibe.ch.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Clémençon
authors.affiliation: Institute of Biochemistry and Molecular Medicine (IBMM), National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Fine
authors.affiliation: Institute of Biochemistry and Molecular Medicine (IBMM), National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland. Electronic address: matthias.hediger@ibmm.unibe.ch.
authors.forename: Matthias A
authors.initials: MA
authors.lastname: Hediger
mesh_terms: D003902:Detergents / Q000737:chemistry* / Q000494:pharmacology*; D008823:Micelles; D055503:Protein Multimerization / Q000187:drug effects*; D020836:Protein Structure, Quaternary / Q000187:drug effects; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003902:Detergents; D008823:Micelles; D029701:Saccharomyces cerevisiae Proteins; D050995:Voltage-Dependent Anion Channel 1
keywords: ATP binding; Detergent; Membrane protein purification; Membrane protein structure; Micro-scale thermophoresis (MST); Mitochondria; Porin; Transmission electron microscopy; Voltage-dependent anion channel VDAC; X-ray diffraction; β-Barrel membrane protein