Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance.
Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.
Unité Mixte de Recherche 7099, Centre National de Recherche Scientifique and Université Paris 7, Institut de Biologie Physico-Chimique, 13 rue Pierre-et-Marie Curie, F-75005 Paris, France.
Paola
P
Bazzacco
Emmanuelle
E
Billon-Denis
K Shivaji
KS
Sharma
Laurent J
LJ
Catoire
Sophie
S
Mary
Christel
C
Le Bon
Elodie
E
Point
Jean-Louis
JL
Banères
Grégory
G
Durand
Francesca
F
Zito
Bernard
B
Pucci
Jean-Luc
JL
Popot
D001436:Bacteriorhodopsins / Q000737:chemistry; D002021:Buffers; D002474:Cell-Free System; D016825:Chlamydomonas reinhardtii / Q000378:metabolism; D045347:Cytochromes b6 / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry; D054439:Ghrelin / Q000737:chemistry; D006031:Glycosylation; D019617:Halobacterium salinarum / Q000378:metabolism; D007477:Ions; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D011437:Propylamines / Q000737:chemistry*; D017510:Protein Folding; D054440:Receptors, Ghrelin / Q000737:chemistry
D002021:Buffers; D045347:Cytochromes b6; D054439:Ghrelin; D007477:Ions; D008565:Membrane Proteins; D011108:Polymers; D011437:Propylamines; D054440:Receptors, Ghrelin; C499017:amphipol A8-35; D001436:Bacteriorhodopsins; D019204:GTP-Binding Proteins