Characterization of linear forms of the circular enterocin AS-48 obtained by limited proteolysis

Abstract 

AS-48 is a 70-residue circular peptide from Enterococcus faecalis with a broad antibacterial activity. Here, we produced by limited proteolysis a protein species carrying a single nicking and fragments of 55 and 38 residues. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that circularization is not required for the bactericidal activity, but it is important to stabilize the native structure. Moreover, it is possible to reduce the sequence to a minimal AS-48 domain without causing inactivation of this bacteriocin.

Abbreviations: CD, circular dichroism, E/S, enzyme to substrate ratio, ESI-MS, electrospray ionization-mass spectrometry, RP, reverse phase, HPLC, high-performance liquid chromatography, [θ], mean residue ellipticity, TFA, trifluoroacetic acid, Tris, tris(hydroxymethyl)aminomethane, SDS, sodium dodecyl sulfate, UV, ultraviolet, TFE, trifluoroethanol, Gdn-HCl, guanidine hydrochloride, MIC, minimum inhibitory concentration, AS_10/11, AS-48 nicked at the level of residues Ala10 and Val11, AS_43–27, fragment 43–27 of AS-48, AS_42,43–10, fragments 42–10 and 43–10 of AS-48

Keywords: Enterocin AS-48, Bacteriocin, Circular protein, Limited proteolysis, Protein fragment, Antimicrobial activity