Solid-State NMR of Viral Fusion Peptides and Proteins

项目主任/首席调查员(最后、第一、中间)：大卫·保罗·韦利基 项目摘要/摘要 拟议研究的长期目标是详细了解病毒融合蛋白的基础- 诱导膜融合。病毒和靶细胞膜的融合是许多病毒感染的关键步骤。 在疾病中的重要性和对融合机制的详细了解将有助于抗病毒的发展 作用模式为融合抑制的治疗学。融合蛋白也是病毒疫苗的靶点。 发展。建议的研究重点是血凝素HA2和gp41融合蛋白。 流感病毒和人类免疫缺陷病毒。之所以选择这些蛋白质是因为这些 病毒会导致人类的主要疾病，因为这些蛋白质是其他I类病毒的原型 融合蛋白。虽然蛋白质序列不是同源的，但这两种蛋白质都是单程的 病毒膜的跨膜蛋白，其~180残基的胞外结构域位于 病毒。特别强调的是~25个残基的N末端融合多肽(FP)结构域，它发挥着一种 在融合催化中起关键作用，被认为在融合过程的早期插入宿主细胞膜。 有四个目标，包括HA2(目标1)和gp41(目标2)的FP的膜结构，以及 HA2(目标3)和gp41(目标4)的FP的膜位置。每个目标分为(A)仅限FP和 (B)FP+可溶性胞外结构域+跨膜结构域切片。两种蛋白质的FP‘s都可以采用任一种α 螺旋或反平行的β片状结构，这两种结构都催化膜融合。该项目的重点是 αHA2和βgp41 FP结构。通过推论，进一步发展了结构/功能相关性 HA2 G1E和gp41 V2E突变导致融合和病毒严重受损的检测 感染。研究FP结构和膜接触的主要技术是固体核磁 共振(SS核磁共振)，特别强调旋转-回波双共振(REDOR)，它可以 阐明原子核间的近似度和距离≤10？REDOR将用于确定螺旋间HA2 FP βgp41FP结构的结构和反平行寄存器。将为以下对象确定部分总体 不同的结构，以及结构和种群如何随着融合受损突变而变化。 将对FP/膜接触进行更详细的测量，以及补充SS核磁共振 用顺磁松弛和1H自旋扩散测量FP/水接触 OMB编号0925-0001/0002(03/16修订版批准至2018年10月31日)页面续格式页面

Program Director/Principal Investigator (Last, First, Middle): Weliky, David Paul Project Summary/Abstract The long-term goal of the proposed research is detailed understanding of 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 will aid development of anti-viral therapeutics whose mode of action is fusion inhibition. Fusion proteins are also a target of viral vaccine development. The proposed research focuses on the hemagglutinin HA2 and gp41 fusion proteins of the influenza virus and human immunodeficiency virus, respectively. These proteins are chosen because these viruses cause major human diseases and because the proteins serve as prototypes for other class I viral fusion proteins. Although the protein sequences are non-homologous, both proteins are single-pass transmembrane proteins of the viral membrane with ~180-residue ectodomain regions which lie outside the virus. There is particular emphasis on the ~25-residue N-terminal “fusion peptide” (FP) domain which plays a key role in fusion catalysis and is thought to insert into the host cell membrane early in the fusion process. There are four Aims which include structures in membrane of the FP's of HA2(Aim 1), and gp41(Aim 2), and the membrane locations of the FP's of HA2(Aim 3) and gp41(Aim 4). Each Aim is divided into (a) FP-only and (b) FP+soluble ectodomain+transmembrane domain sections. The FP's of both proteins can adopt either α helical or antiparallel β sheet structure, and both structures catalyze membrane fusion. The project focuses on the α HA2 and β gp41 FP structures. Structure/function correlations are further developed with corollary measurements on HA2 G1E and gp41 V2E mutants which both result in greatly impaired fusion and viral infection. The main technique to elucidate FP structure and membrane contacts is solid-state nuclear magnetic resonance(SSNMR) with particular emphasis on rotational-echo double resonance(REDOR) which can elucidate internuclear proximities and distances ≤10Å. REDOR will be used to determine interhelical HA2 FP structures and antiparallel registries of β gp41 FP structures. Fractional populations will be determined for the different structures as well as how the structures and populations change with fusion-impairing mutations. There will be REDOR measurements of FP/membrane contacts as well as complementary SSNMR measurements of FP/water contacts using paramagnetic relaxation and 1H spin diffusion. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page