Abstract
Amphipols are a class of novel surfactants that are capable of stabilizing the native state of membrane proteins. They have been shown to be highly effective, in some cases more so than detergent micelles, at maintaining the structural integrity of membrane proteins in solution, and have shown promise as vehicles for delivering native membrane proteins into the gas phase for structural interrogation. Here, we use fast photochemical oxidation of proteins (FPOP), which irreversibly labels the side chains of solvent-accessible residues with hydroxyl radicals generated by laser photolysis of hydrogen peroxide, to compare the solvent accessibility of the outer membrane protein OmpT when solubilized with the amphipol A8-35 or with n-dodecyl-β-maltoside (DDM) detergent micelles. Using quantitative mass spectrometry analyses, we show that fast photochemical oxidation reveals differences in the extent of solvent accessibility of residues between the A8-35 and DDM solubilized states, providing a rationale for the increased stability of membrane proteins solubilized with amphipol compared with detergent micelles, as a result of additional intermolecular contacts.Graphical ᅟ
Highlights
D espite the broad array of essential functions executed by membrane proteins (MPs), high resolution structural data for this class of proteins are lacking compared with their water-soluble counterparts
fast photochemical oxidation of proteins (FPOP) labeling of a protein can result in a number of covalent chemical modifications [24, 25, 32], with the most commonly encountered being the addition of an oxygen atom, accompanied by a mass increase of 16 Da
It can be seen that FPOP oxidation of a protein can result in a small but reproducible decrease in the retention time of an oxidized tryptic peptide compared with its unmodified counterpart when using reverse-phase chromatography: Figure 2a shows the retention time window within which peptide T4 elutes (15-16 mins), comparing the control experiment with the FPOP experiment
Summary
D espite the broad array of essential functions executed by membrane proteins (MPs), high resolution structural data for this class of proteins are lacking compared with their water-soluble counterparts. The favorable attributes of FPOP include the fast labeling times and the irreversible nature of the chemical modifications, the latter permitting comprehensive downstream analysis using LC-MS/MS methods Following such labeling experiments, the proteins can be subjected to proteolysis and the resulting peptides separated and sequenced using LC-MS/ MS (Figure 1). We apply FPOP-LC-MS/MS to gain insights into the interaction sites of two types of amphiphiles, the detergent DDM and the amphipol A8-35, with the 35.3 kDa β-barrel outer membrane protein OmpT. This protein comprises 10 β-strands, with both intermembrane and extramembrane regions, and functions in vivo as an endopeptidase [2, 8, 28, 29]. MP:amphipol complexes have been shown to be more stable over time than their detergent solubilized
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