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.
Although many periplasmic folding factors have been identified, the mechanisms by which they interact with unfolded outer membrane proteins (OMPs) to promote correct folding and membrane insertion remain poorly understood. Here, we have investigated the effect of two chaperones, Skp and SurA, on the folding kinetics of the OMP, PagP. Folding kinetics of PagP into both zwitterionic diC12:0PC (1,2-dilauroyl-sn-glycero-3-phosphocholine) liposomes and negatively charged 80:20 diC12:0PC:diC12:0PG [1,2-dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol)] liposomes were investigated using a combination of spectroscopic and SDS-PAGE assays. The results indicate that Skp modulates the observed rate of PagP folding in a manner that is dependent on the composition of the membrane and the ionic strength of the buffer used. These data suggest that electrostatic interactions play an important role in Skp-assisted substrate delivery to the membrane. In contrast, SurA showed no effect on the observed folding rates of PagP, consistent with the view that these chaperones act by distinct mechanisms in partially redundant parallel chaperone pathways that facilitate OMP assembly. In addition to delivery of the substrate protein to the membrane, the ability of Skp to prevent OMP aggregation was investigated. The results show that folding and membrane insertion of PagP can be restored, in part, by Skp in conditions that strongly favour PagP aggregation. These results illustrate the utility of in vitro systems for dissecting the complex folding environment encountered by OMPs in the periplasm and demonstrate the key role of Skp in holding aggregation-prone OMPs prior to their direct or indirect delivery to the membrane.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.