title: IR-spectroscopic characterization of an elongated OmpG mutant.
abstract: OmpG is a nonselective, pH dependent outer membrane protein from Escherichia coli. It consists of 281 residues, forming a 14-stranded β-sheet structure. In this study, OmpG is extended by 38 amino acids to produce a 16-stranded β-barrel (OmpG-16S). The resulting protein is investigated by IR-spectroscopy. The secondary structure, pH-dependent opening/closing mechanism, buffer accessibility and thermal stability of OmpG-16S are compared to OmpG-WT. The results show that OmpG-16S is responsive to pH change as indicated by the Amide I band shift upon a switch from acidic to neutral pH. This spectral shift is consistent with that observed in OmpG-WT, which confirms the existence of structural differences consistent with the presence of the open or closed state. Secondary structure analysis after curve-fitting of Amide I band revealed that the additional residues do not fold into β-sheet; rather they are in the form of turns and unordered structure. In thermal stability experiments, OmpG-16S is found to be as stable as OmpG-WT. Additionally, H/D exchange experiments showed no difference in the exchange rate of OmpG-16S between the acidic and alkaline pH, suggesting that the loop L6 is no longer sufficient to block the pore entrance at acidic pH.
authors.affiliation: Atilim University, Physics Unit, Biophysics Laboratory, 06836 Golbasi, Ankara, Turkey. Electronic address: filiz.korkmaz@atilim.edu.tr.
authors.forename: Filiz
authors.initials: F
authors.lastname: Korkmaz
authors.affiliation: Max Planck Institute for Biophysics, Department of Structural Biology, Max-von-Laue Str. 3, D-60438 Frankfurt am Main, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: van Pee
authors.affiliation: Max Planck Institute for Biophysics, Department of Structural Biology, Max-von-Laue Str. 3, D-60438 Frankfurt am Main, Germany.
authors.forename: Özkan
authors.initials: Ö
authors.lastname: Yildiz
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D018272:Porins / Q000737:chemistry* / Q000235:genetics*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C114347:OmpG protein, E coli; D018272:Porins
keywords: IR spectroscopy; Outer membrane protein (OMP); Porin; Protein engineering; β-Barrel