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Structure-function studies of defensins and tryptophan-rich antimicrobial peptides.

机译：防御素和富含色氨酸的抗菌肽的结构功能研究。

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摘要

This thesis has focused on the study of two classes of antimicrobial peptides found in vertebrates, the tryptophan-rich antimicrobial peptides (tritrpticin, indolicidin and lactoferricin B (LfcinB)) and the human defensins. The Trp-rich cationic antimicrobial peptides were observed to preferentially insert into anionic phospholipid bilayers, becoming localized to the interfacial region of the bilayer. Using solution NMR spectroscopy, the three-dimensional structures of tritrpticin and a hexa-peptide fragment of LfcinB, thought to be its antimicrobial-active center, were determined in membrane-mimetic SDS micelles. Additionally, a structure-function study was performed on seven analogues of tritrpticin, in which various amino acids were altered. The interactions between the peptides and membranes were characterized using various fluorescence assays, with comparisons being made to their antimicrobial activities, hemolytic activities and their three-dimensional structures in detergent micelles. The research on tryptophan-rich antimicrobial peptides expanded to include the Tip-rich membrane-proximal region of HIV gp41, postulated to disrupt lipid packing during the fusion of the viral and host-cell membranes. The NMR solution structure of a 19-residue peptide corresponding to this region was determined in dodecylphosphocholine micelles. The peptide adopts a helical structure, with the seven aromatic residues forming a collar around the axial length of the peptide that would likely position this peptide in the membrane-water interface of a phospholipid bilayer. Due to the capacity of the Trp-rich peptide to induce lysis of phospholipid vesicles, structure-function studies of this peptide were initiated with the goal of converting the peptide into an antimicrobial peptide. Defensins are 3--5 kDa, cationic antimicrobial peptides with three disulfide bonds. The NMR structure and oligomeric state of human beta-defensin-3 (HBD-3) in aqueous solution was determined and compared to the NMR structures of two other related beta-defensins. While a possible dimer interface was proposed for HBD-3, using homonuclear NMR techniques it was not possible to solve its dimeric structure. Therefore, synthetic genes were designed for HBD-3 and HNP-1 (an alpha-defensin that is also dimeric). proHNP-1, mature-HNP-1 and mature-HBD-3 were successfully expressed to high levels as thioredoxin fusion proteins. Their expression in the OrigamiB strain of E. coli resulted in the successful oxidation of three disulfide bonds.
机译：本论文着重研究在脊椎动物中发现的两类抗菌肽，即富含色氨酸的抗菌肽（三色肽，吲哚米定和乳铁蛋白B（LfcinB））和人防御素。观察到富含Trp的阳离子抗微生物肽优先插入阴离子磷脂双层中，并局限于双层的界面区域。使用溶液NMR光谱法，在模拟膜的SDS胶束中确定了曲美汀和LfcinB的六肽片段的三维结构（被认为是其抗菌活性中心）。另外，对三苯甲基蝶呤的七个类似物进行了结构功能研究，其中多种氨基酸被改变。肽和膜之间的相互作用使用各种荧光测定法进行了表征，并对其在洗涤剂胶束中的抗菌活性，溶血活性和三维结构进行了比较。富含色氨酸的抗菌肽的研究扩展到包括HIV gp41的富含尖端的膜区域，被认为在病毒膜和宿主细胞膜融合期间破坏脂质堆积。在十二烷基磷酸胆碱胶束中确定了与该区域相对应的19个残基肽的NMR溶液结构。该肽采用螺旋结构，七个芳香族残基在肽的轴向长度周围形成一个环，这很可能将该肽置于磷脂双层的膜-水界面中。由于富含Trp的肽具有诱导磷脂囊泡裂解的能力，因此开始了对该肽的结构功能研究，目的是将其转化为抗菌肽。防御素是具有三个二硫键的3--5 kDa阳离子抗菌肽。测定了水溶液中人β-防御素3（HBD-3）的NMR结构和低聚状态，并将其与其他两种相关的β-防御素的NMR结构进行了比较。虽然提出了可能的HBD-3二聚体界面，但使用同核NMR技术无法解决其二聚体结构。因此，为HBD-3和HNP-1（也是一种二聚体的α-防御素）设计了合成基因。 proHNP-1，成熟的HNP-1和成熟的HBD-3作为硫氧还蛋白融合蛋白成功地高表达。它们在大肠杆菌的OrigamiB菌株中的表达导致三个二硫键的成功氧化。

著录项

  • 作者

    Schibli, David John.;

  • 作者单位

    University of Calgary (Canada).;

  • 授予单位 University of Calgary (Canada).;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2003
  • 页码 351 p.
  • 总页数 351
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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