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令人惊讶地常见，并执行多种功能，如免疫防御、药物递送和离子通道门控。然而，阳离子残基，特别是精氨酸，如何克服自由能屏障插入到脂质膜的疏水部分知之甚少。阳离子膜蛋白的结构测定落后于疏水膜蛋白，从而限制了我们对许多健康相关的细胞过程，如先天免疫和离子通道门控的理解。这项工作的广泛，长期的目标是阐明插入和跨脂质膜的阳离子蛋白质易位的结构基础。一般的方法是使用高分辨率固态NMR光谱来确定阳离子膜蛋白的原子级结构和膜拓扑结构、插入深度和取向。直接在脂质膜中探测结构信息的能力和高分辨率魔角旋转NMR提供的位点特异性是我们的结构测定方法的独特优势。我们建议研究两种哺乳动物来源的抗菌肽和一种细胞穿透肽，它们都富含精氨酸。基于我们在最后一个资助期的发现，我们假设胍盐阳离子通过与脂质磷酸盐阴离子络合插入脂质膜的疏水部分，这样做会产生暂时或永久的膜缺陷。我们将通过1）对2-折叠抗菌肽PG-1的Arg-去除、Arg-改变和Arg-二甲基化变体的结构和脂质相互作用进行比较研究，以评估电荷-电荷吸引相对于氢键对磷酸胍络合和PG-1的最终膜破坏活性的相对重要性; 2）确定人抗微生物肽人1-防御素-1在膜中的完整三维结构，阐明其插入深度和取向，并鉴定Arg残基在膜中的位置; 3）研究穿膜肽penetratin的结构和作用机制，并与我们以前研究的抗菌肽的作用机制进行比较。这项研究将采用各种先进的固态NMR技术，如多维相关技术，1H和19 F自旋扩散，核间距离测量，顺磁弛豫增强，和宏观对齐膜的31 P线形。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，以及更好的药物输送化合物来穿过细胞膜。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。它将为设计新的抗菌药物提供更好的结构基础，以作为无耐药性的强效抗生素。这也将有利于开发更有效的药物输送化合物，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.