From in Silico Prediction to in Actu Characterization of a Novel Transmembrane β-Barrel

Abstract

Transmembrane β-barrels (TMBB) constitute a special structural class of proteins localized to the external membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Because TMBBs are surface-accessible proteins that perform a variety of functions ranging from nutrient transport to cellular adhesion, they are tempting targets for vaccine or drug therapy development. Since it could be advantageous to identify TMBB-encoding genes and traditional experimental approaches such as crystallography have proven difficult, computational methods have been explored for identifying genes which encode members of this protein class. However, the cryptic nature of the proteins’sequence-structure relationship has made the computational prediction of TMBB-encoding genes a challenging task. The Freeman-Wimley algorithm, which was developed to predict TMBB-encoding genes from genomic databases, is a highly accurate prediction method based on the physicochemical properties of experimentally characterized TMBB structures. Predicted outer membrane protein L (OmpL) from Salmonella typhimurium LT2, was tested as a model for validating the prediction method. All of the physicochemical and spectroscopic properties exhibited by OmpL are consistent with other known TMBBs. Recombinant OmpL localizes to the outer membrane when expressed in Escherichia coli; OmpL has β-sheet-rich secondary structure with stable tertiary contacts in the presence of either detergent micelles or a lipid bilayer; when reconstituted into a synthetic lipid bilayer, OmpL forms a pore through which small non-electrolyte solutes can diffuse. Together, this data proves that OmpL is a true TMBB and thus, substantiates predictions made by the Freeman-Wimley algorithm.