TY - JOUR
T1 - Conservation of the oligomeric state of native VDAC1 in detergent micelles
AU - Clémençon, Benjamin
AU - Fine, Michael
AU - Hediger, Matthias A.
N1 - Funding Information:
We are grateful to NanoTemper Technologies GmbH for making the MonoLith NT115 device available to our studies and specifically to Dr. François-Xavier Ogi from NanoTemper for technical support and experimental advice and guidance. Also, we thank the group of Prof. Henning Stahlberg, particularly Alexandra Graff-Meyer, for teaching BC in the technique of micro-dialysis. We wish to acknowledge the laboratory of Dr. Gérard Brandolin in CEA Grenoble, DSV, iRTSV, Laboratory of Biochemistry and Biophysics of Integrated Systems where BC performed his PhD thesis on VDAC. Especially, we are grateful to the advice of the excellent scientists in this laboratory with regard to general membrane protein expertise, continuous support, and fruitful discussions. BC and MF wishes to acknowledge the FP7 European Marie Curie Actions International Fellowship Grant, called IFP TransCure (see www.transcure.org ), which financially supported this research. BC also thanks the Rhone Alps funding for supporting earlier work on VDAC in CEA Grenoble.
Publisher Copyright:
© 2016 The Author(s).
PY - 2016/8/1
Y1 - 2016/8/1
N2 - 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.
AB - 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.
KW - ATP binding
KW - Detergent
KW - Membrane protein purification
KW - Membrane protein structure
KW - Micro-scale thermophoresis (MST)
KW - Mitochondria
KW - Porin
KW - Transmission electron microscopy
KW - Voltage-dependent anion channel VDAC
KW - X-ray diffraction
KW - β-Barrel membrane protein
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U2 - 10.1016/j.biochi.2016.05.015
DO - 10.1016/j.biochi.2016.05.015
M3 - Article
C2 - 27238246
AN - SCOPUS:84973131050
SN - 0300-9084
VL - 127
SP - 163
EP - 172
JO - Biochimie
JF - Biochimie
ER -