Investigating lipid transporter protein and lipid interactions using variable temperature electrospray ionization, ultraviolet photodissociation mass spectrometry, and collision cross section analysis

28 May 2024, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Gram-negative bacteria develop and exhibit resistance to antibiotics owing to their highly asymmetric outer membrane maintained by a group of six proteins comprising the Mla (maintenance of lipid asymmetry) pathway. Here we investigate the lipid binding preferences of one Mla protein, MlaC, which transports lipids through the periplasm. We used ultraviolet photodissociation (UVPD) to identify and characterize modifications of lipids endogenously bound to MlaC expressed in three different bacteria strains. UVPD was also used to localize lipid binding to MlaC residues 130-140, consistent with the crystal structure reported for lipid-bound MlaC. The impact of removing the bound lipid from MlaC on its structure was monitored based on collision cross section measurements, revealing that the protein unfolded prior to release of the lipid. The lipid selectivity of MlaC was evaluated based on titrimetric experiments, indicating that MlaC bound lipids in various classes (sphingolipids, glycerophospholipids, fatty acids) as long as they possessed no more than two acyl chains.

Keywords

lipid-protein interactions
ultraviolet photodissociation
native mass spectrometry

Supplementary materials

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Supporting information
Description
Structures and masses of lipids, primer used in MlaC constructs, sequences, masses, and bacteria strains for MlaC, raw UVPD spectra, ejected lipid spectra.
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Spreadsheet supporting information
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UVPD fragment identifications ions for Figures S1-S3 and S9-10.
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