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Solution Behavior and Crystallization of Cytochrome bc 1 in the Presence of Amphipols

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Abstract

Detergents classically are used to keep membrane proteins soluble in aqueous solutions, but they tend to destabilize them. This problem can be largely alleviated thanks to the use of amphipols (APols), small amphipathic polymers designed to substitute for detergents. APols adsorb at the surface of the transmembrane region of membrane proteins, keeping them water-soluble while stabilizing them bio-chemically. Membrane protein/APol complexes have proven, however, difficult to crystallize. In this study, the composition and solution properties of complexes formed between mitochondrial cytochrome bc 1 and A8-35, the most extensively used APol to date, have been studied by means of size exclusion chromatography, sucrose gradient sedimentation, and small-angle neutron scattering. Stable, monodisperse preparations of bc 1/A8-35 complexes can be obtained, which, depending on the medium, undergo either repulsive or attractive interactions. Under crystallization conditions, diffracting three-dimensional crystals of A8-35-stabilized cytochrome bc 1 formed, but only in the concomitant presence of APol and detergent.

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Abbreviations

A8-35:

A specific type of amphipol (Tribet et al. 1996)

APol:

Amphipol

AUC:

Analytical ultracentrifugation

BR:

Bacteriorhodopsin

C8E4 :

Tetraethylene glycol monooctyl ether

Cmc:

Critical micellar concentration

CMP:

Contrast match point

DDM:

Dodecyl-β-d-maltoside

EDTA:

Ethylene-diamine-tetra-acetic acid

EM:

Electron microscopy

FAPolNBD :

Fluorescent (NBD-labeled) A8-35

KMES:

2-Morpholino-ethane sulfonic acid potassium salt

MD:

Molecular dynamics

MW:

Molecular weight

MWCO:

Molecular weight cutoff

NBD:

7-nitrobenz-2-oxa-1,3-diazol-4-yl

NMR:

Nuclear magnetic resonance

βog:

Octyl-β-d-glucopyranoside

OmpX:

Outer membrane protein X from Escherichia coli

PEG4k:

4 kDa polyethylene glycol

SANS and SAXS:

Small-angle neutron and X-ray scattering, respectively

SEC:

Size exclusion chromatography

SERCA1a:

Sarcoplasmic reticulum calcium pump from fast twitch muscle

tOmpA:

Transmembrane domain of outer membrane protein A from E. coli

Tris:

Tris-hydroxymethyl-amino-methane

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Acknowledgments

Particular thanks are due to A.-N. Galatanu and J.-C. Courant for participating in some of these experiments, to P. Hervé for the synthesis of [3H]A8-35, to P. Timmins for his participation in the SANS measurements, to D. Picot and L. Barucq for testing crystals at the ESRF, to I. Gallay for help with the X-ray equipment at the IBPC, to J. Barra and L.J. Catoire for their precious help with the figures, and to Y. Gohon and M. Zoonens for useful comments on the manuscript. This study was supported by the Centre National de la Recherche Scientifique, Université Paris-7, the Human Frontier Science Program Organization (Grant RG00223-2000-M), E.U. Specific Targeted Research Project LSHG-CT-2005-513770 IMPS (Innovative tools for membrane protein structural proteomics), and NIH grant R01DK44842. The stays of EAB in France were subsidized by awards from the France-Berkeley Fund and the French Ministère de l’Education Nationale et de la Recherche.

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Correspondence to Martin Picard.

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Research article destined to the special issue of J. Membr. Biol. on amphipols.

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Charvolin, D., Picard, M., Huang, LS. et al. Solution Behavior and Crystallization of Cytochrome bc 1 in the Presence of Amphipols. J Membrane Biol 247, 981–996 (2014). https://doi.org/10.1007/s00232-014-9694-4

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