Solid-state NMR studies of antimicrobial peptides

描述（由申请人提供）：抗菌肽是动物先天免疫系统中进化高度保留的成分。它们以高效力和速度杀死广泛的微生物，如细菌，病毒和真菌。它们防止病原体耐药性的显着能力使这些肽成为常规抗生素的可行替代品。本研究的主要目的是阐明抗菌作用的结构基础，以便设计具有强杀微生物活性但对哺乳动物细胞的细胞毒性低的新的和改进的抗生素。 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性，如曲率半径、表面电荷和缺乏胆固醇，这些特性与哺乳动物膜不同。为了验证这一假设，我们将专门确定两个代表性的肽与脂质双层，模拟细菌，逆转录病毒和人类红细胞膜的相互作用;确定这些肽在这些不同的脂质膜的取向拓扑结构;并测量这些肽的二级结构和聚集状态。选择的肽是保护蛋白-1（PG-1）和恒河猴θ-防御素1（RTD-1），它们都具有二硫键稳定的β-折叠构象，这是大量抗微生物肽所共有的，包括在人类中发现的防御素。 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，将确定抗菌选择性中的重要脂质因素。 31 P和2 H NMR将用作脂质-肽相互作用的主要探针。关于肽在脂质双层中的取向的信息对于理解肽是否通过孔形成或通过胶束化破坏细胞膜是重要的。这些信息将通过使用定向和未定向静态样品的13 C和15 N NMR实验获得。使用未取向的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由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推进我们对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的知识空白，β-折叠肽如何与脂质双层一般相互作用。

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.