本研究的目的是通过使用色氨酸荧光作为探针,详细表征儿茶素PA-1及其片段与靶膜的结合。基于三维模型(Y.陈,R.Shapira,M.爱森斯坦,还有T.J.Montville,应用。环境。Microbiol.63:524-531,1997),选择了四个合成的N末端儿茶素片段来研究细菌素与膜结合的初始步骤的机制。儿基素PA-1与磷脂酰甘油囊泡的结合,李斯特菌膜的主要成分,导致固有色氨酸荧光强度增加,发射最大值蓝移。缓冲液中和脂质囊泡中的pediacinPA-1荧光的丙烯酰胺猝灭的Stern-Volmer常数分别为8.83+/-0.42和3.53+/-0.67M-1,表明色氨酸残基插入到脂质双层的疏水核心中。当存在带正电荷的氨基酸残基(K-11和H-12)的补丁时,合成的儿茶素片段与脂质囊泡牢固结合,但当该补丁突变时结合较弱。色氨酸荧光参数变化的定量比较,以及山地霉素PA-1及其片段的解离常数,揭示了与脂质囊泡的相对亲和力与肽中的净正电荷平行。含有YGNGV共有基序的片段的相对亲和力比含有阳性片的片段低10倍。此外,将pH值从6.0更改为8.0降低了含有阳性斑块的片段的结合,可能是由于他的残留物去质子化。这些结果表明,静电相互作用,但不是YGNGV主题,控制与目标膜的结合。
The purpose of this study was to characterize in detail the binding of pediocin PA-1 and its fragments to target membranes by using tryptophan fluorescence as a probe. Based on a three-dimensional model (Y. Chen, R. Shapira, M. Eisenstein, and T. J. Montville, Appl. Environ. Microbiol. 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 vesicles of phosphatidylglycerol, the major component of Listeria membranes, caused an increase in the intrinsic tryptophan fluorescence intensity with a blue shift of the emission maximum. The Stern-Volmer constants 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 into the hydrophobic core of the lipid bilayer. The synthetic pediocin fragments bound strongly to the lipid vesicles when a patch of positively charged amino acid residues (K-11 and H-12) was present but bound weakly when this patch was mutated out. Quantitative comparison of changes in tryptophan fluorescence parameters, as well as the dissociation constants for pediocin PA-1 and its fragments, revealed that the relative 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 containing 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 demonstrate that electrostatic interactions, but not the YGNGV motif, govern pediocin binding to the target membrane.