Solid-State NMR of Viral Fusion Peptides and Proteins

这项拟议研究的长期目标是以原子分辨率详细了解 病毒融合蛋白诱导的膜融合。病毒和靶细胞膜的融合是 感染许多在疾病中很重要的病毒，并详细了解融合机制 帮助开发作用模式为融合抑制的抗病毒疗法。拟议的研究 重点介绍人类免疫缺陷病毒(Hiv)的gp41和血凝素ha2融合蛋白。 和流感病毒(IFV)，因为由这些病毒引起的疾病的重要性 因为这些蛋白是其他I类病毒融合蛋白的原型。的确有 特别强调融合蛋白的“融合肽”(FP)结构域，它代表~20-残基 蛋白质N-末端的非极区。大量的生化和生物物理研究表明 FP在融合中起关键作用，化学合成的肽与FP序列可以 作为有用的模型系统来理解病毒/靶细胞融合的某些方面。 长期目标将通过四个具体目标来实现。其中两个目标侧重于固态硬盘 化学交联型二聚体和三聚体HIV的核磁共振结构分析 膜中的FPS和洗涤剂胶束中这些多肽的额外液态核磁共振研究。这个 这些C末端交联肽的寡聚体拓扑反映了FPS在体内的预期拓扑结构 HIV gp41融合蛋白。第三个目标侧重于：(A)确定FPS的插入方向 使用膜在堆叠的玻璃板之间取向的样品的膜；和(B) 用~(13)C核与~(31)P核磁共振距离测量探测~(31)P插入深度 脂类中有19F核。第四个具体目标是SS核磁共振结构和插入深度 含有FP的HIV和IFV大融合蛋白结构域的测量。。

The long-term goal of the proposed research is to understand with atomic-resolution detail the basis for viral fusion protein-induced membrane fusion. Fusion of viral and target cell membranes is a key step in infection for many viruses important in disease and a detailed understanding of fusion mechanisms should aid development of anti-viral therapeutics whose mode of action is fusion inhibition. The proposed research focuses on the gp41 and the hemagglutinin HA2 fusion proteins of the human immunodeficiency virus (HIV) and the influenza virus (IFV), respectively, because of the significance of the diseases caused by these viruses and because these proteins serve as prototypes for other class I viral fusion proteins. There is particular emphasis on the "fusion peptide" (FP) domains of the fusion proteins, which represent ~20-residue apolar regions at the N-termini of the proteins. Numerous biochemical and biophysical studies have shown that the FP plays a key role in fusion and that chemically synthesized peptides with the.FP sequence can serve as useful model systems to understand some aspects of viral/target cell fusion. The long-term objectives will be achieved with four specific aims. Two of the aims focus on solid-state nuclear magnetic resonance (SSNMR) structural analysis of chemically cross-linked dimeric and trimeric HIV FPs in membranes and additional liquid-state NMR studies of these peptides in detergent micelles. The oligomeric topology of these C-terminal cross-linked peptides reflects the expected topology of FPs in the HIV gp41 fusion protein. The third aim focuses on: (a) determination of the insertion orientation of FPs in membranes using samples in which the membranes are oriented between stacked glass plates; and (b) probing FP insertion depths using SSNMR distance measurements between 13C nuclei in the FP and 31P and 19F nuclei in the lipid. The fourth specific aim focuses on SSNMR structural and insertion depth measurements for large HIV and IFV fusion protein domains which contain FPs. .