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常见，具有多种功能，如免疫防御、药物输送和离子通道门控。然而，阳离子残基，特别是精氨酸，如何克服自由能障碍，插入到脂膜的疏水部分，目前还知之甚少。阳离子膜蛋白的结构测定滞后于疏水性膜蛋白，从而限制了我们对许多与健康相关的细胞过程的了解，如先天免疫和离子通道门控。这项工作的广泛、长期的目标是阐明阳离子蛋白跨脂膜插入和转移的结构基础。通常的方法是用高分辨固体核磁共振波谱来确定阳离子膜蛋白的原子级结构和膜的拓扑结构、插入深度和取向。直接探测脂膜结构信息的能力和高分辨率魔角旋转核磁共振提供的位置特异性是我们结构确定方法的独特优势。我们建议研究两种哺乳动物来源的抗菌肽和一种细胞穿透肽，它们都富含Arg。根据我们在上一次授权期中的发现，我们假设鸟粪离子通过与脂质磷酸阴离子络合进入脂膜的疏水部分，从而产生暂时性或永久性的膜缺陷。我们将通过1)对2页抗菌肽PG-1的精氨酸去除、精氨酸修饰和精氨酸二甲基化变异体的结构和脂质相互作用的比较研究来验证这一假设，以评估电荷吸引和氢键对磷酸鸟苷络合和最终对PG-1的膜破坏活性的相对重要性；2)确定人类抗菌肽人1-防御素-1在膜中的完整三维结构，阐明其插入深度和定向，并确定Arg残基在膜中的位置；3)研究细胞穿透性多肽的结构和作用机制，并与我们以前研究的抗菌多肽的作用机制进行比较。这项研究将使用各种先进的固体核磁共振技术，如多维相关技术、1H和19F自旋扩散、核间距离测量、顺磁驰豫增强和宏观取向膜的31P线型。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，并帮助更好地将药物输送到细胞膜上。公共卫生相关性：拟议的研究在两个领域具有广泛的健康相关性。这将为设计新的抗菌剂作为有效的抗生素提供更好的结构基础。这也将有利于开发更有效的药物传递化合物，在不损害细胞膜完整性的情况下穿过细胞膜。

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.