Parasin I, a 19-aa antimicrobial peptide isolated from the catfish skin, shows potent antimicrobial activity against a broad spectrum of pathogens. Therefore, to elucidate the structural features that are required for its potent antimicrobial activity and to produce parasin I analogs with increased activity, we studied the relationship between the structure and the activity of parasin I and investigated the mechanism of action of parasin I. The structure of parasin I, determined by nuclear magnetic resonance analysis, consisted of an alpha-helical region (residues 9-17) flanked by random coil regions (residues 1-8 and 18-19). To elucidate the structure-activity relationship of parasin I, we synthesized a series of N- and C-terminally truncated or amino acid-substituted synthetic parasin I analogs and examined their antimicrobial activity. Deletion of the lysine residue at the N-terminal abolished the antimicrobial activity whereas the deletion of the C-terminal random coil region slightly increased the antimicrobial potency of the peptide. Progressive deletions at the C-terminal did not cause substantial changes in antimicrobial activity of the resulting peptides until five residues were truncated whereupon the antimicrobial activity was almost completely destroyed. Regardless of helical content, a basic residue at the N-terminal was necessary for the peptides to bind to Escherichia coli cell membranes. Among the membrane-binding peptides, only those peptides with helical structure were able to permeabilize the cell membranes. Our results suggest that the lysine residue at the N-terminal mediates the binding of parasin I to the cell membrane while the helical structure of parasin I is responsible for the membrane permeabilizing activity, which kills the microorganism. A systematic approach to overcome the salt-sensitivity of parasin I was designed by the application of helix-capping motifs. The effect of helix-capping motifs on salt-sensitive antimicrobial peptides was further studied using model helical peptides. The helix-capping motifs, APKAM and LQKKGI, were chosen to stabilize the model alpha-helical antimicrobial peptides, which consisted of [RLLR] or [KLLK] repeats, under high salt concentrations. The alpha-helical content of the model peptides was almost completely destroyed at salt concentrations over 200 mM NaCl and this structural change caused 8~32-fold decrease in antimicrobial activity. However, the introduction of helix-capping motifs, APKAM (N) and LQKKGI (C), into the N- and C-terminal of the model alpha-helical sequences, respectively, resulted in structurally stable peptides $(N-[RLLR]_2-C and N-[KLLK]2-C)$, which, upon exposure to 200 mM NaCl, did not lose alpha-helical content and retained antimicrobial activity. Confocal fluorescence microscopic analysis and membrane permeabilization assays showed that $N-[RLLR]_2-C$ and $N-[KLLK]_2-C$ localized, regardless of salt concentrations, to the cell membrane and permeabilized both the outer and cytoplasmic membranes whereas $[RLLR]_n$ and $[KLLK]_n$ (n=3~5) failed to bind to the E. coli cell membrane at high salt concentrations. Our results suggest that structural instability, not hindered electrostatic interaction between the positively-charged peptide and the negatively-charged bacterial cell membranes, is responsible for salt-sensitivity of antimicrobial peptides and that the adoption of helix-capping motifs into salt-sensitive antimicrobial peptides can provide the necessary structural stability for the peptides to retain alpha-helical conformation, thereby enabling the peptides to bind and permeabilize the microbial cell membranes causing cell death, under physiological concentrations.
파라신 I의 truncation 및 substitution 유도체들을 합성하여 이들의 항균력, 구조, 및 작용기작을 분석하여 파라신 I의 구조-활성 관계와 작용기작을 규명하였다. 파라신 I의 N-말단에 위치한 라이신은 파라신이균 세포막에 binding하는데 있어서 중요한 역할을 하는 것을 확인하였으며 helical 구조가 있어야지만 세포막을 permeabilize 시킬 수 있는 것을 규명하였다. 또한 helix-capping motif의 도입으로 파라신 I을 salt-resistant하게 만들어 염 농도에 상관없이 강한 항균력을 나타나게 하였다. Helix-capping motif가 구조를 안정화시켜 salt-resistance를 항균펩타이드에 부여하는 원리를 다른 helical 펩타이드들에서도 확인하였다.
이러한 결과들은 보다 강한 항균력을 갖는 항균 펩타이드 개발에 응용할 수 있으며 항균 펩타이드 작용기작을 더 자세히 규명할 수 있는 이론적인 바탕을 제공하게 될 것이다.
