Amphipathic polymers enable the study of functional membrane proteins in the gas phase.
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.
Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Aneika C
AC
Leney
Lindsay M
LM
McMorran
Sheena E
SE
Radford
Alison E
AE
Ashcroft
D000217:Acyltransferases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005740:Gases; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism; D018272:Porins / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005740:Gases; D011108:Polymers; D018272:Porins; C405564:ompT protein, bacteria; D000217:Acyltransferases; C468520:PagP protein, E coli