Solid-state NMR of antimicrobial and cationic membrane peptides

描述（由申请人提供）：阳离子膜蛋白在自然界中非常普遍，具有多种功能，如免疫防御，药物传递和离子通道门控。然而，阳离子残基，尤其是精氨酸，是如何克服自由能屏障，插入到脂质膜的疏水部分的，目前还不清楚。阳离子膜蛋白的结构测定落后于疏水性膜蛋白，因此限制了我们对许多与健康相关的细胞过程的理解，如先天免疫和离子通道门控。这项工作的广泛、长期目标是阐明阳离子蛋白在脂质膜上插入和易位的结构基础。一般的方法是利用高分辨率固体核磁共振光谱来确定阳离子膜蛋白的原子水平结构和膜拓扑结构、插入深度和取向。高分辨率魔角自旋核磁共振成像直接探测脂膜结构信息的能力和位点特异性是我们的结构测定方法的独特优势。我们建议研究两种来自哺乳动物的抗菌肽和一种细胞穿透素肽，它们都富含精氨酸。根据我们在上一个拨款期的发现；我们假设，胍离子通过与脂质磷酸阴离子络合进入脂质膜的疏水部分，从而产生短暂或永久的膜缺陷。我们将通过1)对2片抗菌肽PG-1的结构和脂质相互作用进行比较研究，以评估电荷吸引与氢键对胍-磷酸络合和PG-1最终的膜破坏活性的相对重要性；2)确定了人1-防御素-1抗菌肽在膜中的完整三维结构，阐明了其插入深度和取向，并确定了Arg残基在膜中的位置；3)研究细胞穿透肽——穿透素的结构和作用机制，并与我们之前研究的抗菌肽的作用机制进行比较。本研究将采用多种先进的固态核磁共振技术，如多维相关技术、1H和19F自旋扩散、核间距离测量、顺磁弛豫增强和宏观排列膜的31P线形状。由此产生的结构信息将有助于设计更有效的抗生素来对抗细菌耐药性和更好的跨细胞膜药物递送化合物。公共卫生相关性：拟议的研究在两个领域具有广泛的卫生相关性。这将为设计新的抗微生物药物提供更好的结构基础，使其成为无耐药性的强效抗生素。它还将有利于开发更有效的药物传递化合物，这些化合物可以穿过细胞膜而不破坏其完整性。

DESCRIPTION (provided by applicant): Cationic membrane proteins are surprisingly common in nature and carry out a wide variety of functions such as immune defense, drug delivery, and ion channel gating. Yet how cationic residues, especially Arg, overcome the free-energy barrier to insert into the hydrophobic part of the lipid membrane is poorly understood. Structure determination of cationic membrane proteins has lagged behind that of hydrophobic membrane proteins, thus limiting our understanding of many health-related cellular processes such as innate immunity and ion channel gating. The broad, long-term objective of this work is to elucidate the structural basis for the insertion and translocation of cationic proteins across lipid membranes. The general approach is to determine the atomic-level structure and the membrane topology depth of insertion and orientation of cationic membrane proteins using high-resolution solid-state NMR spectroscopy. The ability to probe structural information directly in the lipid membrane and the site specificity afforded by high-resolution magic-angle spinning NMR are unique advantages of our structure determination approach. We propose to investigate two antimicrobial peptides of mammalian origin and a cell-penetratin peptide, all rich in Arg. Based on our finding in the last grant period; we hypothesize that guanidinium cations insert into the hydrophobic part of the lipid membrane by complexing with the lipid phosphate anions, in so doing creating either transient or permanent membrane defects. We will test this hypothesis by 1) a comparative study of the structure and lipid-interaction of Arg-removed, Arg-altered, and Arg- dimethylated variants of the 2-sheet antimicrobial peptide PG-1, to assess the relative importance of charge-charge attraction versus hydrogen bonding to guanidinium-phosphate complexation and to the eventual membrane-disruptive activity of PG-1; 2) determining the complete three-dimensional structure of a human antimicrobial peptide, human 1-defensin-1, in the membrane, elucidating its depth of insertion and orientation, and identifying the location of Arg residues in the membrane; 3) investigating the structure and mechanism of action of a cell-penetrating peptide, penetratin, to compare with the mechanisms of antimicrobial peptides we studied before. This research will employ a wide variety of advanced solid-state NMR techniques, such as multidimensional correlation techniques, 1H and 19F spin diffusion, internuclear distance measurements, paramagnetic relaxation enhancement, and 31P lineshapes of macroscopically aligned membranes. The resulting structural information will help the design of more potent antibiotics to combat bacterial resistance and better drug-delivery compounds to cross cell membranes. PUBLIC HEALTH RELEVANCE: The proposed research has broad health relevance in two areas. It will provide a better structural basis for designing new antimicrobial agents to serve as potent antibiotics without resistance. It will also benefit the development of more effective drug-delivery compounds that cross the cell membrane without damaging its integrity.