Solid-State NMR of Viral Fusion Peptides and Proteins

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免疫治疗的关键步骤。 感染的许多病毒的重要疾病和详细了解融合机制， 有助于开发其作用方式为融合抑制的抗病毒治疗剂。拟议研究 重点关注人类免疫缺陷病毒（HIV）的gp41和血凝素HA2融合蛋白 和流感病毒（IFV），分别，因为由这些引起的疾病的意义 病毒，并且因为这些蛋白质充当其它I类病毒融合蛋白的原型。有 特别强调融合蛋白的"融合肽"（FP）结构域，其代表约20个残基 在蛋白质的N-末端的非极性区域。大量的生物化学和生物物理学研究表明， FP在融合中起关键作用，化学合成的具有FP序列的肽可以 作为有用的模型系统来理解病毒/靶细胞融合的某些方面。 将通过四个具体目标实现长期目标。其中两个目标集中在固态 化学交联的二聚体和三聚体HIV的核磁共振（SSNMR）结构分析 膜中的FP和这些肽在洗涤剂胶束中的额外液态NMR研究。的 这些C-末端交联肽的寡聚拓扑结构反映了FP的预期拓扑结构， HIV gp41融合蛋白。第三个目标集中于：（a）确定FP在细胞中的插入取向。 使用样品的膜，其中膜在堆叠的玻璃板之间取向;和（B） 使用FP中的13C核和31P核之间的SSNMR距离测量来探测FP插入深度 和脂质中的19F核。第四个具体目标侧重于SSNMR结构和插入深度 测量含有FP的大HIV和IFV融合蛋白结构域。.

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. .