Solid-state NMR studies of antimicrobial peptides

说明(申请人提供)：抗菌肽是进化过程中高度保存的动物先天免疫系统成分。它们以高效和快速的方式杀死细菌、病毒和真菌等广泛的微生物。它们非凡的防止病原耐药性的能力使这些多肽成为传统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，从而设计出对哺乳动物细胞具有较强杀菌活性但细胞毒性较低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，这些机制源于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的膜不同。为了验证这一假设，我们将专门确定两个具有代表性的多肽与模拟细菌、逆转录病毒和人类红细胞膜的脂双层的相互作用；确定这些多肽在这些不同的脂膜中的定向拓扑；并测量这些多肽的二级结构和聚集状态。选择的多肽是前列腺素-1(PG-1)和恒河猴Theta-Defensin 1(RTD-1)，它们都具有二硫键稳定的β-折叠构象，这与许多抗菌肽一样，包括在人类中发现的防御素。 我们将使用一种集成的固态核磁共振方法来研究这两种β-折叠多肽的作用机制。通过研究具有特定膜曲率、胆固醇含量和阴离子表面电荷的脂质中的脂肽相互作用，将确定影响抗微生物选择性的重要脂类因素。31P和2H核磁共振将作为脂-肽相互作用的主要探针。关于脂双层中肽方向的信息对于了解多肽是通过孔形成还是通过胶束化破坏细胞膜是重要的。这一信息将通过使用定向和非定向静态样品的13C和15N核磁共振实验获得。使用无取向样品提取分子取向的能力将使我们能够测量多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核磁共振各向同性化学位移和多量子实验来确定。总之，与脂膜特性相关的新的结构信息将极大地促进我们对β-折叠抗菌肽作用机制的理解。此外，这项拟议的研究将填补我们关于β-折叠多肽如何与一般脂双层相互作用的知识空白。

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.