ELECTROSTATIC INTERACTIONS, BUT NOT THE YGNGV CONSENSUS MOTIF, GOVERNTHE BINDING OF PEDIOCIN PA-1 AND ITS FRAGMENTS TO PHOSPHOLIPID-VESICLES

The purpose of this study was to characterize in detail the binding of pediocin PA-1 and its fragments to target membranes by using tryptoph an fluorescence as a probe. Based on a three-dimensional model (Y. Che n, R. Shapira, M. Eisenstein, and T. J. Montville, Appl. Environ. Micr obiol. 63:524-531, 1997), four synthetic N-terminal pediocin fragments were selected to study the mechanism of the initial step by which the bacteriocin associates with membranes. Binding of pediocin PA-1 to ve sicles of phosphatidylglycerol, the major component of Listeria membra nes, caused an increase in the intrinsic tryptophan fluorescence inten sity with a blue shift of the emission maximum. The Stern-Volmer const ants for acrylamide quenching of the fluorescence of pediocin PA-1 in buffer and in the lipid vesicles were 8.83 +/- 0.42 and 3.53 +/- 0.67 M-1, respectively, suggesting that the tryptophan residues inserted in to the hydrophobic core of the lipid bilayer. The synthetic pediocin f ragments bound strongly to the lipid vesicles when a patch of positive ly charged amino acid residues (K-11 and H-12) was present but bound w eakly when this patch was mutated out. Quantitative comparison of chan ges in tryptophan fluorescence parameters, as well as the dissociation constants for pediocin PA-1 and its fragments, revealed that the rela tive affinity to the lipid vesicles paralleled the net positive charge in the peptide. The relative affinity for the fragment containing the YGNGV consensus motif was 10-fold lower than that for the fragment co ntaining the positive patch. Furthermore, changing the pH from 6.0 to 8.0 decreased binding of the fragments containing the positive patch, probably due to deprotonation of His residues. These results demonstra te that electrostatic interactions, but not the YGNGV motif, govern pe diocin binding to the target membrane.