Characterization of the surface layer protein from Azotobacter vinelandii

Abstract

The regular surface array protein (S protein) of Azotobacter vinelandii was extracted from outer membrane fragments with distilled water and further purified by gel filtration chromatography. The protein was shown to behave anomalously on sodium dodecyl sulfate polyacrylamide gels producing a number of conformational isomers. The amino acid composition of S protein was similar to that of other surface array proteins, particularly in its lack of cysteine. The theoretical monomelic molecular weight of S protein was calculated to be 60 218 based on the total amino acid composition and the apparent molecular weight determined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Circular dichroism spectra indicated that S protein was composed of approximately 35% β sheet structure, negligible α helix, with the remainder of the polypeptide backbone aperiodic in nature. The effect of the divalent cations Ca2+ and Mg2+ on the conformation of S protein was examined by circular dichroism spectroscopy; however, no conformational change was detected in response to the presence of these species nor did S-protein monomer aggregate into multimers in the presence of these cations. Purified S-protein monomer was inactive in divalent cation mediated reassembly of the S layer onto the surface of distilled water washed cells. A larger multimeric form present only in fresh preparations appeared to be the active species involved in in vitro reassembly of the A. vinelandii surface array.