Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry

Abstract

Membrane proteins reside in lipid bilayers and are typically extracted from this environment for study, which often compromises their integrity. Here we eject intact assemblies from membranes, without chemical disruption, and use mass spectrometry to define their composition. From E. coli outer membranes, we identify a chaperone-porin association and lipid interactions in the beta-barrel assembly machinery. Bridging inner and outer membranes we observe efflux pumps, and from inner membranes a pentameric pore of TonB, and the protein-conducting channel Sec YEG, in association with F1FO ATP-synthase. Intact mitochondrial membranes from Bos taurus yield respiratory complexes and fatty acid-bound dimers of the ADP/ATP transporter (ANT-1). These results highlight the importance of native membrane environments for retaining small-molecule binding, subunit interactions and associated chaperones of the membrane proteome.

Structure and function of stator units of the bacterial flagellar motor

“…The stoichiometry of ExbBD was uncertain and different experiments reported 4:1, 4:2, 5:2 and 6:3 stoichiometries (42)(43)(44). However, a recent high-resolution structure of ExbBD shows a clear 5:2 stoichiometry (45), which is consistent with the existence of ExbB pentamers in the native Escherichia coli membrane (46).…”

Quantifying the heterogeneity of macromolecular machines by mass photometry

“…Given that different oligomeric complexes vary in molecular mass, mass measurement could in principle be ideally suited to examine sample heterogeneity. Despite the advances in native mass spectrometry (MS) over the past decades [13][14][15] , the associated experimental complexity and non-native conditions have prevented native MS from becoming a widely used tool in this context. Mass photometry (MP), originally introduced as interferometric scattering mass spectrometry (iSCAMS), is a label-free approach that accurately measures molecular mass by quantifying light scattering from single biomolecules in solution 16,17 .…”

Elucidating the Structure of Membrane Proteins

“…Not only did these protein machines then emerge intact, but the analysis also enabled the discovery of their methods of communication, how they find their final location in the membrane and how they transport their molecular luggage into a cell [8]. …”