Sulfonated amphipols: Synthesis, properties, and applications

Abstract

Amphipols (APols) are amphiphatic polymers that keep membrane proteins (MPs) water-soluble. The best characterized and most widely used APol to date, A8-35, comprises a polyacrylate backbone grafted with octyl- and isopropylamine side chains. The nature of its hydrophilic moieties prevents its use at the slightly acidic pH that is desirable to slow down the rate of amide proton exchange in solution NMR studies. We describe here the synthesis and properties of pH-insensitive APols obtained by replacing isopropyles with taurine. Sulfonated APols (SAPols) can be used to trap MPs in the form of small complexes, to stabilize them, and to keep them water-soluble even at low pH. [(15) N,(1) H]-transverse relaxation-optimized spectroscopy NMR spectra obtained at pH 6.8 of a bacterial outer MP folded in SAPols show that the protein is correctly folded. The spectra have a resolution similar to that achieved with A8-35 and reveal water-exposed amide and indole protons whose resonance peaks are absent at pH 8.0.

Labeling and Functionalizing Amphipols for Biological Applications

“…The latter, when all of them are ionized, i.e., at pH ≥ 7, make A8-35 highly water soluble (>200 g L −1 ), whereas the octyl chains make it amphipathic. The isopropyl groups limit the charge density, which, if it is too high, seems to negatively affect MP stability (Bazzacco et al 2012; Dahmane et al 2011; Picard et al 2006), without contributing much to the global hydrophobicity at low or ambient temperature (Takei et al 1993). …”

“…If isopropylamine is replaced with taurine, the end product is a sulfonated APol (‘SAPol’) (Fig. 7) (Dahmane et al 2011; Picard et al 2006). SAPol cannot be synthesized by HMPAS because of the poor solubility of taurine in NMP.…”

“…APols are relatively large molecules: the number-average molar mass of the most common APol, called A8-35 (see below), is ~4.3 kDa (Giusti et al 2014b), that of more recently developed phosphocholine-based (Diab et al 2007a, b), sulfonated (Dahmane et al 2011; Picard et al 2006) or glycosylated, non-ionic (Bazzacco et al 2012; Sharma et al 2012) APols similar or larger. APols assemble into small, globular, micelle-like particles, with, in the case of A8-35, a particle mass of ~40 kDa (Gohon et al 2004, 2006), that is, on average, ~9 molecules.…”

Folding and stability of integral membrane proteins in amphipols

“…76 An unsuccessful attempt to fold a b-barrel in solution has been 77 reported for the b-barrel domain of AIDA [32]. However even for 78 AIDA, folding was successful when the protein was bound to a 79 nickel-bearing column [33]. 80 a-Helical TMPs like BR or KcsA have been folded from dena- 81 tured forms in organic solvent like trifluoroacetic acid or mixtures 82 of formic acid and ethanol [2,11].…”

“…Other APols developed more recently 151 include glycosylated nonionic APols (NAPols) [77,78] [50,51,73]). SAPols [79] are derived from a precursor of A8-35 lack- 155 ing the amidation with isopropylamine. Instead, about $40% of the 156 carboxyl groups are amidated by taurine (2-aminoethanesulfonic 157 acid) (Fig.…”