Solid-state NMR studies of antimicrobial peptides

抗菌肽的固态核磁共振研究

• 批准号: 6560666
• 负责人: Mei Hong
• 金额: $ 22.58万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6560666
• 关键词: antibiotics; antiinfective agents; cell membrane; cholesterol; defensins; high performance liquid chromatography; immunity; lipid bilayer membrane; mass spectrometry; microorganism; nuclear magnetic resonance spectroscopy; peptides

DESCRIPTION (provided by applicant): Antimicrobial peptides are evolutionarily highly preserved elements of the innate immune system in animals. They kill a wide range of microbial organisms such as bacteria, viruses, and fungi with high potency and speed. Their remarkable ability to prevent pathogenic resistance makes these peptides a viable alternative to conventional antibiotics. The broad objective of this research is to elucidate the structural basis for antimicrobial action, so that new and improved antibiotics with strong microbiocidal activity but low cytotoxicity to mammalian cells may be designed. The central hypothesis of this project is that antimicrobial peptides share common mechanisms of action that derive from the special characteristics of microbial membranes such as radius of curvature, surface charge, and lack of cholesterol, characteristics that are distinct from mammalian membranes. To test this hypothesis, we will specifically determine the interactions of two representative peptides with lipid bilayers that mimic bacterial, retroviral, and human erythrocyte membranes; determine the orientation topology of these peptides in these various lipid membranes; and measure the secondary structure and aggregation state of these peptides. The peptides of choice are protegrin-1 (PG-1) and rhesus theta-defensin 1 (RTD-1), which both possess a disulfide-bond stabilized beta-sheet conformation that is common to a large number of antimicrobial peptides, including the defensins found in humans. We will use an integrated solid-state nuclear magnetic resonance (NMR) approach to study the mechanism of action of these two beta-sheet peptides. The important lipid factors in antimicrobial selectivity will be identified by studying the lipid-peptide interactions in lipids with defined membrane curvature, cholesterol content, and anionic surface charges. 31P and 2H NMR will be used as the main probes for the lipid-peptide interaction. Information on the peptide orientation in the lipid bilayer is important for understanding whether the peptides disrupt the cell membrane by pore formation or by micellization. This information will be obtained by 13C and 15N NMR experiments using both oriented and unoriented static samples. The ability to extract molecular orientation using unoriented samples will allow us to measure the concentration-dependence and membrane-curvature-dependence of the peptide orientation. The secondary structure and aggregation of PG-1 and RTD-1 in lipid bilayers will be determined from NMR isotropic chemical shifts and multiple-quantum experiments, respectively. Together, the new structural information, correlated with the characteristics of lipid membranes, will significantly advance our understanding of the mechanism of action of beta-sheet antimicrobial peptides. Moreover, the proposed research will fill our knowledge gap of how beta-sheet peptides interact with lipid bilayers in general.

描述（由申请人提供）：抗菌肽是动物先天免疫系统在进化上高度保留的元素。它们能够高效、快速地杀死多种微生物，例如细菌、病毒和真菌。它们具有防止病原体耐药性的卓越能力，使这些肽成为传统抗生素的可行替代品。这项研究的主要目标是阐明抗菌作用的结构基础，以便设计出具有强抗菌活性但对哺乳动物细胞具有低细胞毒性的新型和改进的抗生素。 该项目的中心假设是，抗菌肽具有共同的作用机制，这些机制源自微生物膜的特殊特征，例如曲率半径、表面电荷和不含胆固醇，这些特征与哺乳动物膜不同。为了检验这一假设，我们将专门确定两种代表性肽与模拟细菌、逆转录病毒和人类红细胞膜的脂质双层的相互作用；确定这些肽在这些不同脂膜中的取向拓扑；并测量这些肽的二级结构和聚集状态。选择的肽是 protegrin-1 (PG-1) 和恒河猴 theta-防御素 1 (RTD-1)，它们都具有二硫键稳定的 β-折叠构象，这是大量抗菌肽（包括人类中发现的防御素）所共有的。 我们将使用集成的固态核磁共振（NMR）方法来研究这两种β-折叠肽的作用机制。通过研究具有确定的膜曲率、胆固醇含量和阴离子表面电荷的脂质中的脂质-肽相互作用，可以确定抗菌选择性中重要的脂质因素。 31P和2H NMR将用作脂质-肽相互作用的主要探针。有关脂双层中肽方向的信息对于了解肽是否通过孔形成或胶束化破坏细胞膜非常重要。该信息将通过使用定向和非定向静态样品的 13C 和 15N NMR 实验获得。使用未定向样品提取分子取向的能力将使我们能够测量肽取向的浓度依赖性和膜曲率依赖性。 PG-1 和 RTD-1 在脂质双层中的二级结构和聚集将分别通过 NMR 各向同性化学位移和多量子实验确定。总之，与脂膜特性相关的新结构信息将显着促进我们对 β-折叠抗菌肽作用机制的理解。 此外，拟议的研究将填补我们关于β-折叠肽如何与脂质双层相互作用的知识空白。