Dissociation and reassembly of Escherichia coli type 1 pili.
Escherichia coli type 1 pili, which mediate the mannose-sensitive adherence of the bacterium to eucaryotic cells, are comprised of very stable arrays of pilin protein subunits (molecular weight, approximately 17,000). Previous methods for the dissociation of pili caused their irreversible denaturation. We have found that incubation of pili in saturated guanidine hydrochloride at 37 degrees C led to their complete dissociation, as evidenced by nephelometry and electron microscopy. Gel chromatography of the dissociated pili on a Sepharose CL-6B column in the presence of saturated guanidine hydrochloride yielded a single protein peak with a molecular weight corresponding to that of pilin. Dialysis of this peak against 5 mM tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0) and rechromatography in the same buffer afforded a major protein peak, probably consisting of pilin dimers. About 25% of the protein in this peak bound to a mannan-sepharose column and could be eluted with methyl alpha-D-mannoside. The pilin dimer gave a single protein band upon polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate (molecular weight, 16,600) or 10 M urea and penetrated completely into 7% gels in the absence of denaturants. Reassembly of the pilin dimers into pili was achieved upon dialysis against the tris(hydroxymethyl)aminomethane buffer containing 5 mM MgCl2, as observed by electron microscopy. Thus, the conditions used allow renaturation of the dissociated subunits and may aid in further studies of the structure-function relationship of pili.
D004926:Escherichia coli / Q000032:analysis / Q000648:ultrastructure*; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000032:analysis / Q000648:ultrastructure*; D019791:Guanidine; D006146:Guanidines; D046911:Macromolecular Substances; D008351:Mannans; D008565:Membrane Proteins; D012685:Sepharose
D006146:Guanidines; D046911:Macromolecular Substances; D008351:Mannans; D008565:Membrane Proteins; D036781:Fimbriae Proteins; D012685:Sepharose; D019791:Guanidine