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Updated: May 5, 2026
From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins
Published on: July 4, 2016
Methionine mutations of outer membrane protein X influence structural stability and beta-barrel unfolding
Deepti Chaturvedi1, Radhakrishnan Mahalakshmi
1Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, Madhya Pradesh, India.
Abstract:
We report the biochemical and biophysical characterization of outer membrane protein X (OmpX), an eight-stranded transmembrane β-barrel from E. coli, and compare the barrel behavior with a mutant devoid of methionine residues. Transmembrane outer membrane proteins of bacterial origin are known to display high tolerance to sequence rearrangements and mutations. Our studies with the triple mutant of OmpX that is devoid of all internal methionine residues (M18L; M21L; M118L) indicate that Met replacement has no influence on the refolding efficiency and structural characteristics of the protein. Surprisingly, the conserved substitution of Met→Leu leads to barrel destabilization and causes a lowering of the unfolding free energy by a factor of ∼8.5 kJ/mol, despite the mutations occurring at the loop regions. We report that the barrel destabilization is accompanied by a loss in cooperativity of unfolding in the presence of chemical denaturants. Furthermore, we are able to detect an unfolding intermediate in the Met-less barrel, whereas the parent protein exhibits a classic two-state unfolding. Thermal denaturation measurements also suggest a greater susceptibility of the OmpX barrel to heat, in the Met-less construct. Our studies reveal that even subtle variations in the extra-membrane region of rigid barrel structures such as OmpX, may bear severe implications on barrel stability. We propose that methionines contribute to efficient barrel structuring and protein-lipid interactions, and are therefore important elements of OmpX stability.
Insights
Methionine residues in E. coli outer membrane protein X (OmpX) are crucial for barrel stability. Removing methionine destabilizes the OmpX barrel, impacting protein structure and lipid interactions.
Area of Science:
- Biochemistry
- Biophysics
- Structural Biology
Background:
- Bacterial outer membrane proteins (OMPs) exhibit remarkable sequence tolerance.
- Outer membrane protein X (OmpX) is an eight-stranded transmembrane β-barrel protein from E. coli.
- Methionine residues are often found in OMPs, but their specific role in barrel stability is not fully understood.
Purpose of the Study:
- To biochemically and biophysically characterize OmpX and a mutant lacking methionine residues.
- To investigate the impact of methionine depletion on OmpX refolding, structural characteristics, and stability.
- To elucidate the role of methionine in OmpX barrel structuring and protein-lipid interactions.
Main Methods:
- Site-directed mutagenesis to create a triple mutant of OmpX lacking internal methionine residues (M18L; M21L; M118L).
- Biochemical assays to assess refolding efficiency and structural characteristics.
- Chemical and thermal denaturation experiments to determine unfolding thermodynamics and kinetics.
- Spectroscopic techniques to monitor protein unfolding and detect intermediates.
Main Results:
- Methionine depletion did not affect OmpX refolding efficiency or overall structural characteristics.
- The Met-less OmpX mutant exhibited significant barrel destabilization, with a decrease in unfolding free energy (∼8.5 kJ/mol).
- Unfolding cooperativity was reduced in the Met-less mutant, and an unfolding intermediate was detected, unlike the parent protein's two-state unfolding.
- Thermal denaturation revealed increased heat susceptibility in the Met-less OmpX construct.
- Mutations in loop regions significantly impacted barrel stability, suggesting methionine's role in extra-membrane regions.
Conclusions:
- Methionine residues are critical for maintaining the stability of the OmpX β-barrel structure.
- Subtle variations in extra-membrane regions can have profound effects on rigid barrel protein stability.
- Methionine likely contributes to efficient barrel structuring and favorable protein-lipid interactions, underscoring its importance for OmpX stability.
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