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Topology of PhoE porin: the 'eyelet' region

M Struyvé1, J Visser, H Adriaanse

  • 1Department of Molecular Cell Biology, University of Utrecht, The Netherlands.

Molecular Microbiology
|January 1, 1993
PubMed

Insights

The third loop of PhoE porin is located within the pore channel, not exposed externally. This finding, based on genetic insertions and antibody binding, impacts the porin

Area of Science:

  • Structural biology
  • Microbiology
  • Molecular genetics

Background:

  • A model suggests PhoE porin traverses the outer membrane 16 times, exposing eight loops externally.
  • The location of the third external loop of PhoE has remained undetermined.
  • The structure of Rhodobacter capsulatus porin, similar to PhoE, features an 'eyelet' region within the pore.

Purpose of the Study:

  • To determine the location of the predicted third external loop of PhoE.
  • To investigate if the third loop contributes to an 'eyelet' structure within the porin channel.

Main Methods:

  • Insertion of multiple copies of an oligonucleotide linker encoding a foot-and-mouth disease virus (FMDV) VP1 antigenic determinant into the PhoE gene.
  • Expression of hybrid PhoE proteins and verification of their insertion into the outer membrane.
  • Binding assays using a monoclonal antibody (MA11) against the FMDV epitope.
  • Antibiotic sensitivity tests and single-channel conductance measurements.

Main Results:

  • Hybrid PhoE proteins were correctly inserted into the outer membrane.
  • Monoclonal antibody MA11 binding indicated successful insertion and accessibility of the FMDV epitope.
  • Antibiotic sensitivity and channel conductance measurements showed that insertions altered the channel size.
  • Results suggest the third loop is located within the pore channel, not externally.

Conclusions:

  • The third loop of PhoE is located within the pore channel.
  • This internal location is consistent with the loop forming an 'eyelet' structure, similar to that observed in R. capsulatus porin.
  • The findings provide evidence for the PhoE porin model and its functional implications.

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