Mutant OmpF porins of Yersinia pseudotuberculosis with deletions of external loops: structure-functional and

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O V Sidorova1, V A Khomenko1, O Yu Portnyagina1

  • 1G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok 690022, Russia.

Biochemical and Biophysical Research Communications
|February 18, 2014
PubMed

Insights

Site-directed mutagenesis created Yersinia pseudotuberculosis OmpF porin mutants lacking extracellular loops. These OmpF mutants retained pore-forming activity but showed altered antigenic structure.

Area of Science:

  • Microbiology
  • Structural Biology
  • Immunology

Background:

  • Outer membrane proteins (OMPs) like porins are crucial for bacterial transport and interaction.
  • Yersinia pseudotuberculosis OmpF porin is a key outer membrane protein involved in nutrient uptake and pathogenicity.
  • Understanding the structure-function relationship of OmpF is vital for developing targeted antimicrobial strategies.

Purpose of the Study:

  • To investigate the role of specific extracellular loops (L1, L4, L6, L8) in the structure, function, and antigenicity of Yersinia pseudotuberculosis OmpF porin.
  • To generate and characterize recombinant OmpF mutants with deletions in single extracellular loops.
  • To assess the impact of loop deletions on protein structure, pore activity, and antigenic properties.

Main Methods:

  • Site-directed mutagenesis was employed to create OmpF mutants with single extracellular loop deletions.
  • Proteins were expressed in Escherichia coli and purified from inclusion bodies.
  • Structural analysis utilized SDS-PAGE, circular dichroism, and intrinsic protein fluorescence.
  • Pore-forming activity was assessed using the bilayer lipid membrane technique.
  • Antigenic structure changes were evaluated using enzyme-linked immunosorbent assay (ELISA).

Main Results:

  • Recombinant OmpF mutants were successfully expressed and purified.
  • Secondary and tertiary structures showed some alterations compared to wild-type OmpF.
  • Mutant porins exhibited pore-forming activity identical to the wild-type OmpF.
  • Deletion of extracellular loops significantly altered the antigenic structure of the OmpF porin.

Conclusions:

  • Extracellular loops of Yersinia pseudotuberculosis OmpF porin play a significant role in its antigenic structure.
  • While structural and antigenic properties are affected, the core pore-forming function is maintained upon deletion of these loops.
  • These findings contribute to understanding OmpF structure-function relationships and have implications for vaccine development and diagnostics.
Keywords:
Deletions of the external loopsOmpF porinRecombinant proteinsYersinia pseudotuberculosis

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