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Investigation of Pore Formation in Lipid Bilayer by Antimicrobial Peptides

机译：用抗菌肽研究脂质双层中的孔形成

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摘要

The first part of the study investigated the interaction of multiple melittin peptides with the lipid bilayer. Melittin is a naturally occurring antimicrobial peptide that has the ability to kill bacterial cells through cell membrane penetration leading to pore formation. In this investigation, all atom molecular dynamics (MD) simulation has been carried out to describe the interaction of 2,4 or 6 peptides placed on the surface of 3:1 ratio of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) lipid bilayer (a mimic of bacterial cell membrane) corresponding to protein-lipid (P/L) ratio of 2/96, 4/162 or 6/166 respectively. MD simulation was also carried out for (i) 1-8 transmembrane peptides (corresponding P/L ratio of 1/128 to 8/128) in a 3:1 ratio of DOPC/DOPG mixed membrane for symmetric as well as asymmetric placement of peptides and (ii) 4-8 transmembrane peptides (corresponding to P/L ratio of 4/128 to 8/128) for pure DOPC lipid bilayer (a mimic of mammalian cell membrane) for asymmetric placement of peptides. This study also quantified the potential of mean force through molecular dynamics (MD) simulation for the addition of melittin into DOPC/DOPG mixed bilayer, a mimic of bacterial membrane, for different extents of insertion into either a bilayer or a pore consisting of three to six transmembrane peptides.;The second part of the study proposed a methodology that is based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy beta-conglycinin (7S) subunits. Initial screening of peptide segments from soy beta-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, one peptide (coded as 7a16) satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to initial structure. The antimicrobial activity of this peptide against L. monocytogenes and E. coli was further confirmed by spot-on-lawn test.;The third part of this research studied the interaction between curcumin (CUR) and lipid bilayer. CUR is a widely used natural food ingredient with known ability of targeting microorganism cell membrane, but the detailed mechanism remains unclear. In this study, the interaction of CUR with different types of model lipid bilayer (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)), mixture of model lipid bilayer (POPE/POPG with ratio of 3:1), and realistic biological membranes (E. coli and yeast) were investigated by all atom explicit solvent molecular dynamics (MD) simulation over microseconds. CUR readily inserts into different types of model lipid bilayer system in liquid state within the first two hundred nanoseconds of the simulation, staying in the lipid tails region, near the interface of lipid head and lipid tail. Parallel orientation to the membrane surface is found to be more probable than perpendicular orientation for CUR as indicated by the tilt angle distribution. This orientation preference is less significant as the fraction of POPE is increased in the system, likely due to the better water solvation of perpendicular orientation in POPE bilayer. In E. coli and yeast bilayers, tilt angle distributions were similar to that for POPE/POPG mixed bilayer. Water hydration number around CUR in these more realistic bilayers is higher than the corresponding value for POPE/POPG bilayer. In this part, antimicrobial activities of CUR against L. monocytogenes and E. coli were validated using bioassay, viability assay, transmission electron microscopy (TEM), and fluorescence dye leakage experiments. CUR exhibited antimicrobial activity when dissolved in dimethylformamide (DMF).;The last part of the study compared the fluorescence dye leakage of 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) / cholesterol liposome induced by melittin and its mutants. The results indicated: (i) dye leakage is dependent on the concentration of peptide, whereas a critical peptide concentration is needed for pore formation, (ii) a lag time is needed for pore formation above the critical peptide concentration, which implies that pore formation occurred by nucleation, and (iii) the changes of fluorescence intensity are consistent with the antimicrobial activities of these peptides. This technique not only captures the nucleation of pore formation, but also describes the event of growth of pore size. In addition, the balance between net charge and hydrophobicity is essential for design of appropriate pore forming peptides. (Abstract shortened by ProQuest.).
机译：研究的第一部分研究了多种蜂毒肽与脂质双层的相互作用。蜂毒肽是一种天然存在的抗菌肽，具有通过细胞膜渗透导致孔形成而杀死细菌细胞的能力。在这项研究中，已进行了所有原子分子动力学（MD）模拟，以描述置于3：1比的1,2-二油酰基-sn-甘油-3-磷酸胆碱表面上的2,4或6个肽的相互作用（DOPC）和1,2-二-（9Z-十八烯酰基）-sn-甘油-3-磷酸-（1'-rac-甘油）（DOPG）脂质双层（细菌细胞膜的模拟物），对应于蛋白质-脂质（P / L）比率分别为2 / 96、4 / 162或6/166。还对DOPC / DOPG混合膜的3：1比例的（i）1-8个跨膜肽（对应的P / L比为1/128至8/128）进行了MD模拟，以对称和不对称地放置肽和（ii）4-8个跨膜肽（对应于4/128至8/128的P / L比）用于纯DOPC脂质双层（模拟哺乳动物细胞膜），用于不对称放置肽。这项研究还通过分子动力学（MD）仿真定量了将蜂毒蛋白添加到DOPC / DOPG混合双层（细菌膜的模拟物）中的平均力的潜力，以不同程度插入双层或由三到三层组成的孔六种跨膜肽。研究的第二部分提出了一种基于对肽-脂双层相互作用进行机械评估的方法，以从大豆β-伴大豆球蛋白（7S）亚基中鉴定AMP。从大豆β-伴大豆球蛋白（7S）亚基中初步筛选肽段是基于其疏水性，疏水力矩和净电荷。亲水和疏水相互作用之间的微妙平衡对于孔的形成是必要的。高疏水性降低了肽在水相中的溶解度，而高亲水性限制了肽与双层的结合。从最初的筛选中选择的几种候选物中，一种肽（编码为7a16）满足抗菌活性的标准，即。 （i）脂质-肽在表面状态下结合，（ii）跨膜状态下的聚集体孔形成。这种通过分子动力学模拟鉴定抗菌活性的方法被证明是可靠的，因为它对初始结构不敏感。草坪斑点试验进一步证实了该肽对单核细胞增生李斯特菌和大肠杆菌的抗菌活性。第三部分研究姜黄素（CUR）与脂质双层的相互作用。 CUR是一种广泛使用的天然食品成分，具有靶向微生物细胞膜的已知能力，但具体机制尚不清楚。在这项研究中，CUR与不同类型的模型脂质双层（1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸乙醇胺（POPE），1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸甘油的相互作用） （POPG），1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱（POPC），1,2-二醇酰基-sn-甘油-3-磷酸胆碱（DOPC）和1,2-二棕榈酰基-sn-甘油通过所有原子显式溶剂分子动力学（MD）模拟研究了-3-磷酸乙醇胺（DPPE），模型脂质双层混合物（POPE / POPG的比例为3：1）和实际的生物膜（大肠杆菌和酵母菌）超过微秒。在模拟的前两百纳秒内，CUR可以很容易地以液态插入到不同类型的模型脂质双层系统中，停留在脂质尾巴区域中，靠近脂质头和脂质尾巴的界面。如倾斜角分布所示，发现与膜表面平行的取向比垂直取向的CUR更有可能。随着系统中POPE分数的增加，这种取向偏好的重要性降低了，这可能是由于POPE双层中垂直取向的更好的水溶剂化所致。在大肠杆菌和酵母双层膜中，倾斜角分布与POPE / POPG混合双层膜的倾斜角分布相似。在这些更逼真的双层中，CUR附近的水合数高于POPE / POPG双层的相应值。在这一部分中，使用生物测定，生存力测定，透射电子显微镜（TEM）和荧光染料渗漏实验验证了CUR对单核细胞增生李斯特菌和大肠杆菌的抗菌活性。当溶解在二甲基甲酰胺（DMF）中时，CUR表现出抗菌活性。研究的最后一部分比较了蜂毒肽及其突变体诱导的1,2-二肉豆蔻酰基-sn-甘油-3-磷酸胆碱（DMPC）/胆固醇脂质体的荧光染料泄漏。结果表明：（i）染料泄漏取决于肽的浓度，而孔形成需要临界肽浓度，（ii）高于临界肽浓度的孔形成需要一定的滞后时间，这意味着孔形成通过成核发生（iii）荧光强度的变化与这些肽的抗菌活性一致。该技术不仅捕获了孔形成的成核作用，而且还描​​述了孔径增大的事件。此外，净电荷和疏水性之间的平衡对于设计合适的成孔肽至关重要。 （摘要由ProQuest缩短。）。

著录项

  • 作者

    Lyu, Yuan.;

  • 作者单位

    Purdue University.;

  • 授予单位 Purdue University.;
  • 学科 Bioengineering.
  • 学位 Ph.D.
  • 年度 2017
  • 页码 319 p.
  • 总页数 319
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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