Elucidation of the Three-Dimensional Structure of an Antimicrobial Peptide, SakP(N24C + 44C), in a Membrane-Mimicking Environment by the use of NMR Spectroscopy

Abstract

The antimicrobial peptide, sakP(N24C + 44C) is a mutant of a class IIa bacteriocin, sakacin P, produced by lactic acid bacteria. SakP(N24C + 44C) consists of 44 residues and possesses a C-terminal disulphide bond that ties the C-terminal tail to the mid part of the peptide-sequence, in contrast to the wild-type peptide which lacks a C-terminal disulphide bond. The three-dimensional structure of sakP(N24C + 44C) in a membrane-mimicking environment (dodecylphosphocholine (DPC) micelles) was investigated by circular dichroism (CD) and by two-dimensional homo nuclear resonance spectroscopy (NMR). CD spectra indicated that sakP(N24C + 44C) exists as a random coil in water, whereas NMR results indicate that sakP(N24C + 44C) has a well-defined structure in DPC micelles. Simulated annealing calculations based on distance restraints derived from NMR spectra, were used to generate structures of sakP(N24C + 44C). The structures revealed an amphipathic central á-helix (residues 18-33), a less well-defined â-sheet-like structure in the N-terminal half (residues 1-17), and a defined structured C-terminal half (residues 34 -44) without any common secondary structural motif. Comparison of sakP(N24C + 44C) with leucocin A, another pediocin-like (class IIa) bacteriocin, revealed a striking resemblance, which may indicate that these two bacteriocins have the same mode of action on their target cells. The structural features of sakP(N24C + 44C) and their possible role in an antimicrobial mechanism are discussed.

In the lack of a rapid large-scale purification procedure for the purification of sakP(N24C + 44C) that was needed in this study, a new and simple two-step purification procedure for pediocin-like bacteriocins and other cationic peptides was developed. The new procedure yields more than 80% of the activity that is initially in the culture supernatant, and the final bacteriocin preparation is more than 90% pure. With the new purification procedure, it is possible to purify milligram quantities of pediocin-like bacteriocins within a few hours.