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 beta-sheet structure. In this study, OmpG is extended by 38 amino acids to produce a 16-stranded beta-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 beta-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. (C) 2015 Elsevier Inc. All rights reserved.