title: Engineering of an E. coli outer membrane protein FhuA with increased channel diameter.
abstract: BACKGROUND
  Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering.
  
  
  RESULTS
  The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD) spectroscopy shows a high β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine) were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%.
  
  
  CONCLUSIONS
  In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.
authors.affiliation: Department of Biotechnology (Biology VI), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
authors.forename: Manuel
authors.initials: M
authors.lastname: Krewinkel
authors.forename: Tamara
authors.initials: T
authors.lastname: Dworeck
authors.forename: Marco
authors.initials: M
authors.lastname: Fioroni
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D001560:Benzidines / Q000378:metabolism; D002942:Circular Dichroism; D004058:Diffusion; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D005818:Genetic Engineering; D006735:Horseradish Peroxidase / Q000378:metabolism; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001560:Benzidines; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D007473:Ion Channels; C021758:3,3',5,5'-tetramethylbenzidine; D006735:Horseradish Peroxidase