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Updated: Jun 20, 2025
Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions
Published on: February 10, 2022
Investigating Lipid Transporter Protein and Lipid Interactions Using Variable Temperature Electrospray Ionization,
Virginia K James1, Bradley J Voss2, Amanda Helms1
1Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States.
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, and fatty acids) as long as they possessed no more than two acyl chains.
Insights
Researchers studied the MlaC protein
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Gram-negative bacteria possess an asymmetric outer membrane crucial for antibiotic resistance.
- The Mla (maintenance of lipid asymmetry) pathway, comprising six proteins, maintains this membrane structure.
- Understanding lipid transport is key to addressing antibiotic resistance.
Purpose of the Study:
- To investigate the lipid-binding preferences of the MlaC protein, a key component of the Mla pathway.
- To characterize the interaction between MlaC and its bound lipids.
- To determine how lipid binding affects MlaC protein structure and function.
Main Methods:
- Ultraviolet photodissociation (UVPD) was employed to identify and characterize endogenously bound lipids on MlaC.
- Collision cross-section measurements were used to monitor structural changes in MlaC upon lipid removal.
- Titrimetric experiments were conducted to evaluate MlaC's lipid selectivity.
Main Results:
- UVPD successfully identified and characterized lipid modifications bound to MlaC across different bacterial strains.
- Lipid binding was localized to MlaC residues 130-140, aligning with existing crystal structure data.
- MlaC undergoes unfolding before releasing its bound lipid, as indicated by collision cross-section measurements.
- MlaC exhibits selectivity for lipids with a maximum of two acyl chains, including sphingolipids, glycerophospholipids, and fatty acids.
Conclusions:
- MlaC plays a critical role in transporting lipids within the periplasm of Gram-negative bacteria.
- The study elucidates MlaC's lipid-binding site and preferences, contributing to understanding membrane asymmetry maintenance.
- The findings provide insights into the structural dynamics of MlaC during lipid transport and release, potentially informing strategies against antibiotic resistance.
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