Nonionic Homopolymeric Amphipols: Application to Membrane Protein Folding, Cell-Free Synthesis, and Solution Nuclear Magnetic Resonance

Abstract

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.

Mass spectrometry-enabled structural biology of membrane proteins

“…This study revealed additional intermolecular contacts with the amphipol (Fig. 9), consistent with the known abilities of amphipols to stabilize MPs compared with detergents [219,220]. Ligand binding [221], conformational dynamics [222] and conformational changes [223,224] of MPs can also be studied using synchrotron radiolysis HRFP.…”

Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy

“…The Flag-V2R was purified by immunochromatography using an M1 Flag antibody affinity resin (Fig. 1A) and reconstituted by exchanging detergents for NAPols (1:10 protein/ NAPol weight ratio), which are known to enhance the stability of membrane proteins (20). To obtain a homogeneous fraction of the V2R, the NAPol-reconstituted receptors were then loaded onto a size exclusion chromatography column.…”

Membrane Protein Production for Structural Analysis

“…The most classical extracts from E. coli and wheat germ are used routinely for structural approaches as they tolerate additives for the cotranslational solubilization of CF system-expressed IMPs. The palette of molecules for protein solubilization in CF systems is now very varied and is increased by the possibility of compounds mixture (Park et al 2011;Bazzacco et al 2012;Junge et al 2010;Ma et al 2011). A valid alternative to tensioactifs is the insertion in lipid bilayer or nanodiscs (Periasamy et al 2012;Roos et al 2012).…”