Structural investigations on novel porin, OmpAb from Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen and known to cause nosocomial infections especially in ICUs of hospitals. We have previously reported that the novel outer membrane protein, OmpAb from Acinetobacter baumannii is a transmembrane porin and plays an important role in transport of small molecules, like antibiotics across the membrane. In the present study we report the N-terminal sequence, Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis of OmpAb and structural investigations using UV-Vis absorption, circular dichroism (CD), and fluorescence on OmpAb. SDS-PAGE results suggest that OmpAb is actually a heat modifiable monomer and is of 37 kDa at room temperature. Secondary structure of OmpAb is being done for the first time that showed predominantly beta-sheet structure (68%), a feature characteristic of porins. Using N-Bromosuccinimide (NBS) as oxidizing agent, the total number of tryptophans in OmpAb is estimated to be four. The present results indicate that out of the four, two tryptophans seem to be located in the integral part of the membrane, perhaps periplasmic/membrane-bound while the other two tryptophans are exposed to the solvent. We followed the fluorescence emission using conventional 280 mn and selective 305 nm excitation (established by us earlier) to explore the environment of four tryptophans in OmpAb. Emission results using selective excitation of 305 nm revealed local conformational changes of those tryptophans which are on the surface. On urea denaturation and pH dependent denaturation there is a loss of P-sheet structure by more than 70%, this is concomitant with the increase in fluorescence intensity and red shift in lambda(max, em). As reflected by CD spectral data, we also found that OmpAb is fairly stable like other porins up to 70 degrees C. As there are no reports on the structural aspects of any outer membrane proteins of Acinetobacter baumannii, results presented here on this novel major porin, OmpAb, will help in understanding the structure-function relationship.