title: Engineering of the E. coli outer membrane protein FhuA to overcome the hydrophobic mismatch in thick polymeric membranes.
abstract: BACKGROUND
  Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein.
  
  
  RESULTS
  To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 β-sheets, prior to the more regular periplasmatic β-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext). The secondary structure prediction and CD spectroscopy indicate the β-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB(1000)-PEG(6000)-PIB(1000) (PIB = polyisobutylene, PEG = polyethyleneglycol) has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine). Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB(1000)-PEG(6000)-PIB(1000) membrane. Furthermore labeling of the Lys-NH(2) groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer.
  
  
  CONCLUSIONS
  Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.
authors.affiliation: Department of Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
authors.forename: Noor
authors.initials: N
authors.lastname: Muhammad
authors.forename: Tamara
authors.initials: T
authors.lastname: Dworeck
authors.forename: Marco
authors.initials: M
authors.lastname: Fioroni
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000138:chemical synthesis*; D001560:Benzidines / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000138:chemical synthesis*; D006735:Horseradish Peroxidase / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008566:Membranes / Q000737:chemistry*; D008969:Molecular Sequence Data; D011090:Polyenes / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D015202:Protein Engineering; D017433:Protein Structure, Secondary
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001560:Benzidines; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D011090:Polyenes; D011108:Polymers; C021758:3,3',5,5'-tetramethylbenzidine; D011092:Polyethylene Glycols; C008967:polyisobutylene; D006735:Horseradish Peroxidase