Conservation of the oligomeric state of native VDAC1 in detergent micelles.

Clemençon, Benjamin; Fine, Michael; Hediger, Matthias (2016). Conservation of the oligomeric state of native VDAC1 in detergent micelles. Biochimie, 127, pp. 163-172. Elsevier Masson SAS 10.1016/j.biochi.2016.05.015

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Clemençon, Benjamin; Fine, Michael and Hediger, Matthias

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

0300-9084

Publisher:

Elsevier Masson SAS

Language:

English

Submitter:

Kevin Marc Rupp

Date Deposited:

02 Aug 2016 14:10

Last Modified:

02 Aug 2016 14:10

Publisher DOI:

10.1016/j.biochi.2016.05.015

PubMed ID:

27238246

Uncontrolled 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

BORIS DOI:

10.7892/boris.84824

URI:

https://boris.unibe.ch/id/eprint/84824

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