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Outer membrane proteins of Methylococcus capsulatus (Bath)
A Fjellbirkeland1, H Kleivdal, C Joergensen
1Department of Molecular Biology, University of Bergen, Bergen High Technology Centre, N-5020 Bergen, Norway. anne.fjellbirkeland@pki.uib.no
Abstract:
Membranes obtained from whole-cell lysates of Methylococcus capsulatus (Bath) were separated by Triton X-100 extraction. The resulting insoluble fraction was enriched in outer membranes as assessed by electron microscopy and by the content of beta-hydroxy palmitic acid and particulate methane monooxygenase. Major proteins with molecular masses of approximately 27, 40, 46, 59, and 66 kDa were detected by SDS-PAGE of the Triton-X-100-insoluble membranes. MopA, MopB, MopC, MopD, and MopE (Methylococcus outer membrane protein) are proposed to designate these proteins. Several of the Mop proteins exhibited heat-modifiable properties in SDS-PAGE and were influenced by the presence of 2-mercaptoethanol in the sample buffer. The 46- and 59-kDa bands migrated as a single high-molecular-mass 95-kDa oligomer under mild denaturing conditions. When reconstituted into black lipid membranes, this oligomer was shown to serve as a channel with an estimated single-channel conductance of 1.4 nS in 1 M KCl.
Insights
Researchers isolated and characterized outer membrane proteins (Mop proteins) from Methylococcus capsulatus (Bath). One Mop oligomer reconstituted into lipid membranes functions as a channel.
Area of Science:
- Microbiology
- Biochemistry
- Membrane Biology
Background:
- Methylococcus capsulatus (Bath) is a methanotrophic bacterium.
- Bacterial outer membranes contain essential proteins involved in transport and structure.
- Understanding these proteins is crucial for elucidating bacterial physiology.
Purpose of the Study:
- To isolate and characterize proteins from the outer membrane of Methylococcus capsulatus (Bath).
- To identify major protein components and their properties.
- To investigate the functional properties of a specific protein oligomer.
Main Methods:
- Triton X-100 extraction of whole-cell lysates.
- SDS-PAGE for protein separation and molecular mass determination.
- Electron microscopy and biochemical assays for membrane fraction enrichment.
- Reconstitution of proteins into black lipid membranes for functional analysis.
Main Results:
- Outer membranes were successfully isolated and enriched.
- Five major Methylococcus outer membrane proteins (MopA-E) with masses 27-66 kDa were identified.
- Mop proteins showed heat-modifiable properties and sensitivity to reducing agents.
- A 95-kDa oligomer formed by 46- and 59-kDa Mop proteins functioned as a channel in lipid bilayers.
Conclusions:
- The study identified key outer membrane proteins in Methylococcus capsulatus.
- A novel protein oligomer with ion channel activity was characterized.
- These findings contribute to understanding the structure and function of bacterial outer membranes.