Amphipathic Polymers Enable the Study of Functional Membrane Proteins in the Gas Phase

Abstract

Membrane proteins are notoriously challenging to analyze using mass spectrometry (MS) because of their insolubility in aqueous solution. Current MS methods for studying intact membrane proteins involve solubilization in detergent. However, detergents can destabilize proteins, leading to protein unfolding and aggregation, or resulting in inactive entities. Amphipathic polymers, termed amphipols, can be used as a substitute for detergents and have been shown to enhance the stability of membrane proteins. Here, we show the utility of amphipols for investigating the structural and functional properties of membrane proteins using electrospray ionization mass spectrometry (ESI-MS). The functional properties of two bacterial outer-membrane β-barrel proteins, OmpT and PagP, in complex with the amphipol A8-35 are demonstrated, and their structural integrities are confirmed in the gas phase using ESI-MS coupled with ion mobility spectrometry (IMS). The data illustrate the power of ESI-IMS-MS in separating distinct populations of amphipathic polymers from the amphipol–membrane complex while maintaining a conformationally “nativelike” membrane protein structure in the gas phase. Together, the data indicate the potential importance and utility of amphipols for the analysis of membrane proteins using MS.

Amphipols for Each Season

“…The bacterial outer membrane enzyme PagP retains phospholipase activity after being trapped in SMALPs (Knowles et al 2009). OmpT and PagP are functional in A8-35 (Leney et al 2012). The transmembrane domain of the bacterial EII mtl mannitol permease performs the transphosphorylation from phosphoenolpyruvate to mannitol more rapidly after trapping in A8-35 than it does in detergent solution (Opačić et al 2014a).…”

“…At the date of this writing, seven α -helical MPs, including six GPCRs, have been folded in vitro using APols, and four β -barrel ones (see Table 7), with typical yields ranging between 60 and >90 %. For refolding from urea, the protocol generally involves diluting the urea-denatured protein into an APol solution, so as to lower the concentration of urea to non-denaturing levels (Dahmane et al 2011; Leney et al 2012; Pocanschi et al 2006, 2013). For refolding from SDS, the most usual procedure is to precipitate the dodecylsulfate as its potassium salt in the presence of APols (Bazzacco et al 2012; Catoire et al 2010a; Dahmane et al 2009, 2013; Pocanschi et al 2006).…”

“…MPs trapped in A8-35 (Bechara et al 2012; Catoire et al 2009; Hopper et al 2013; Leney et al 2012) or in NAPols (Bechara et al 2012) are amenable to mass spectrometry (MS) using either matrix-assisted laser desorption ionization (MALDI) (Bechara et al 2012; Catoire et al 2009) or electron spray ionization (ESI) (Hopper et al 2013; Leney et al 2012) techniques. As a rule, most MPs and subunits can be detected, but there are, however, some exceptions (Bechara et al 2012).…”

“…ESI–MS coupled with ion-mobility spectrometry (IMS) has been used to quantify the proportions of properly folded versus unfolded protein following A8-35-assisted folding of two β -barrel MPs, OmpT and PagP (Leney et al 2012), as well as to compare the mass and dispersity of individual molecules of unlabeled and perdeuterated A8-35 (Giusti et al 2014b). ESI–MS has been applied with limited success to investigating the oligomeric state of A8-35-trapped DAGK, part of the native trimer fragmenting into dimers and monomers (Hopper et al 2013).…”

Mass spectrometry-enabled structural biology of membrane proteins

“…3 c,d) that were successfully refolded directly into A8-35 [110]. Further studies revealed that the monomeric a-helical inner membrane proteins Mhp1 and GalP could also be liberated from amphipol A8-35 [89], and whilst the trimeric E.coli diacylglycerol kinase (DgkA) could be liberated from amphipol A8-35, the dominant species was the monomer (Fig.…”