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The Oms66 (p66) protein is a Borrelia burgdorferi porin
J T Skare1, T A Mirzabekov, E S Shang
1Department of Medical Microbiology and Immunology, Texas A&M University Health Science Center, College Station 77843, USA. jskare@tamu.edu
Abstract:
In this study we report the purification and characterization of a 66-kDa protein, designated Oms66, for outer membrane-spanning 66-kDa protein, that functions as a porin in the outer membrane (OM) of Borrelia burgdorferi. Oms66 was purified by fast-performance liquid chromatography and exhibited an average single-channel conductance of 9.62 +/- 0.37 nS in 1 M KCl, as evidenced by 581 individual insertional events in planar lipid bilayers. Electrophysiological characterization indicated that Oms66 was virtually nonselective between cations and anions and exhibited voltage-dependent closure with multiple substates. The amino acid sequence of tryptic peptides derived from purified Oms66 was identical to the deduced amino acid sequence of p66, a previously described surface-exposed protein of B. burgdorferi. Purified Oms66 was recognized by antiserum specific for p66 and serum from rabbits immune to challenge with virulent B. burgdorferi, indicating that p66 and Oms66 were identical proteins and that Oms66/p66 is an immunogenic protein in infected rabbits. In a methodology that reduces liposomal trapping and nonspecific interactions, native Oms66 was incorporated into liposomes, confirming that Oms66 is an outer membrane-spanning protein. Proteoliposomes containing Oms66 exhibited porin activity nearly identical to that of native, purified Oms66, indicating that reconstituted Oms66 retained native conformation. The use of proteoliposomes reconstituted with Oms66 and other Oms proteins provides an experimental system for determinating the relationship between conformation, protection, and biological function of these molecules.
Insights
We purified and characterized Oms66, a 66-kDa outer membrane protein from Borrelia burgdorferi that functions as a porin. This protein, identical to p66, is immunogenic and retains its native conformation when reconstituted into proteoliposomes.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Borrelia burgdorferi is the causative agent of Lyme disease.
- The outer membrane (OM) of B. burgdorferi contains essential proteins involved in pathogen-host interactions.
- Porins are crucial outer membrane proteins that regulate the passage of molecules.
Purpose of the Study:
- To purify and characterize a 66-kDa outer membrane protein from B. burgdorferi, designated Oms66.
- To determine the function and properties of Oms66 as a porin.
- To confirm the identity and immunogenicity of Oms66/p66.
Main Methods:
- Fast-performance liquid chromatography (FPLC) for protein purification.
- Planar lipid bilayer electrophysiology to assess porin activity and conductance.
- Tryptic peptide sequencing and antiserum recognition to confirm protein identity.
- Proteoliposome reconstitution to evaluate Oms66 in a native-like environment.
Main Results:
- Oms66 was purified and exhibited single-channel conductance characteristic of a porin.
- Electrophysiological studies showed Oms66 is nonselective and voltage-dependent.
- Oms66 was confirmed to be identical to the previously described p66 surface protein.
- Oms66/p66 is immunogenic in rabbits infected with B. burgdorferi.
- Reconstituted Oms66 in proteoliposomes retained porin activity, indicating native conformation.
Conclusions:
- Oms66 is a functional porin in the outer membrane of Borrelia burgdorferi.
- Oms66/p66 is an immunogenic protein, suggesting potential roles in host-pathogen interactions.
- Proteoliposomes containing Oms66 provide a system to study porin structure-function relationships.