Because of their potential as novel antibiotic agents, antimicrobial peptides (AMPs) have generated considerable interest. The mechanism of bacterial toxicity of AMPs often involves the disruption and/or permeabilization of the bacterial membrane; even those that act intracellularly first have to traverse the membrane. In this work we have explored the incorporation of the fluorinated aromatic amino acids fluoro-Phe and fluoro-Tyr into the Trp- and Arg-rich AMP tritrpticin, and investigated their role in the membrane binding properties and the antimicrobial activity of the peptide. Fluorinated peptides were obtained with good yield by recombinant expression of tritrpticin as a calmodulin-fusion protein in Escherichia coli. Cells were grown in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, and the peptides were released by proteolysis from the purified fusion protein. By using SDS micelles, as a simplified model of the bacterial cytoplasmic membrane, we could study the peptide-membrane interactions and the preferred location of individual fluorinated residues in the micelles by ¹⁹F NMR spectroscopy. Solvent-perturbation ¹⁹F NMR measurements revealed that para-fluoro-Phe residues are embedded deeply in the hydrophobic region of the micelles. On the other hand, 3-fluoro-Tyr residues introduced in tritrpticin were located near the surface of the micelles with high solvent exposure, while 2-fluoro-Tyr sidechains were less solvent exposed. In combination with the outcome of determinations of their antimicrobial activity, our ¹⁹F NMR results indicate that the higher solvent exposure of Tyr residues correlates with a decrease of the antimicrobial potency. This different role of Tyr can likely be extended from tritrpticin to other cationic AMPs.

由于其作为新型抗生素的潜力，抗菌肽（AMP）引起了极大的兴趣。AMPs的细菌毒性机制通常涉及细菌膜的破坏和/或透化。甚至那些先在细胞内起作用的细胞也必须穿过膜。在这项工作中，我们探索了将含氟芳香族氨基酸氟-Phe和氟代-Tyr掺入富含Trp和Arg的AMP tritrpticin中，并研究了它们在膜结合特性和肽的抗菌活性中的作用。通过在大肠杆菌中重组表达曲美霉素作为钙调蛋白融合蛋白，获得了高收率的氟化肽。。细胞在草甘膦（一种芳香族氨基酸生物合成抑制剂）的存在下生长，然后通过蛋白水解从纯化的融合蛋白中释放出这些肽。通过使用SDS胶束，作为细菌细胞质膜的简化模型，我们可以通过¹⁹ F NMR光谱研究胶束中的肽-膜相互作用和单个氟化残基的优选位置。溶剂扰动¹⁹F NMR测量表明对氟苯丙氨酸残基深深嵌入胶束的疏水区域。另一方面，引入三苯甲基吡啶的3-氟-Tyr残基位于胶束表面附近，具有较高的溶剂暴露，而2-氟-Tyr侧链的溶剂暴露较少。结合测定其抗菌活性的结果，我们的¹⁹ F NMR结果表明，Tyr残留物的较高溶剂暴露量与抗菌能力的降低有关。Tyr的这种不同作用可能会从三苯甲基吡啶扩展到其他阳离子AMP。