Department of Biophysics, University of Michigan, Ann Arbor, Michigan, USA.
Biophys J. 2010 Jan 20;98(2):248-57. doi: 10.1016/j.bpj.2009.09.060.
A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an alpha-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. (15)N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. (31)P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.
一段 21 个氨基酸残基的肽段 LL7-27（RKSKEKIGKEFKRIVQRIKDF），对应于唯一的人类抗菌肽 LL37 的 7-27 位残基，显示出对微生物（特别是革兰氏阳性菌）的强大活性，但对红细胞没有活性。通过差示扫描量热法、荧光、圆二色性和 NMR 实验，对 LL7-27 的结构、膜取向和靶膜选择性进行了表征。吖啶萘-8-磺酸摄取实验揭示了 LL37 和 LL7-27 引起大肠杆菌外膜扰动的两种不同模式。圆二色性结果表明，在 LL7-27 的情况下，构象转变是由脂质特异性相互作用介导的，而不是 LL37。它与阴离子（但不是两性离子）囊泡结合时折叠成α-螺旋构象，也不会诱导两性离子脂质囊泡中的染料泄漏。差示扫描量热法热图谱显示，LL7-27 完全整合到 DMPC/DMPG（3:1）脂质体中，但在 DMPC 脂质体中诱导富含肽和贫肽的区域。机械排列脂质双层上的 (15)N NMR 实验表明，与全长肽 LL37 一样，肽 LL7-27 接近双层表面取向，表明该肽具有地毯式作用机制。来自含有 LL7-27 的 POPC/POPG（3:1）双层的 (31)P NMR 谱显示脂质双层结构的明显破坏，与肽诱导 POPC/POPG（3:1）囊泡中的染料泄漏的能力一致。胆固醇被证明可以抑制肽引起的脂质双层结构无序。这些结果解释了细菌对 LL7-27 的敏感性和红细胞的抗性，并且可能对膜选择性治疗剂的设计具有意义。
