Solid-State NMR of Viral Fusion Peptides and Proteins

病毒融合肽和蛋白质的固态核磁共振

基本信息

批准号：
9512067
负责人：
David P Weliky
金额：
$ 31.78万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-09-30 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9512067
关键词：
Acquired Immunodeficiency Syndrome Adopted Amino Acid Sequence Binding Catalysis Cell membrane Cells Chemicals Chimeric Proteins Cholesterol Complex Data Detergents Development Diffusion Disease Goals HIV HIV Envelope Protein gp120 HIV vaccine Helix-Turn-Helix Motifs Hemagglutinin Hydrocarbons Hydrophobicity Impairment In Vitro Infection Influenza Integral Membrane Protein Intracellular Membranes Kinetics Label Length Ligation Lipids Location Magnetic Resonance Measurement Membrane Membrane Fusion Membrane Proteins Modeling Mole the mammal Mutation N-terminal Peptides Pharmaceutical Preparations Physiological Play Population Population Distributions Principal Investigator Process Protein Region Proteins Registries Relaxation Research Rotation SNAP receptor Sampling Sequence Homology Signal Transduction Structure Techniques Tertiary Protein Structure Testing Therapeutic Transmembrane Domain Vertebral column Viral Viral Fusion Proteins Viral Vaccines Virus Virus Diseases Water Work base beta pleated sheet comparative design endosome membrane functional disability human disease influenzavirus insight loss of function mimetics monomer mutant novel peptide B peptide chemical synthesis peptide structure preference programs prototype receptor solid state nuclear magnetic resonance vaccine development virus envelope

项目摘要

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

项目总监/首席研究员（最后，第一，中间）：Weliky，David Paul 项目概要/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- 诱导的膜融合是许多病毒感染的关键步骤。在疾病中很重要，对融合机制的详细了解将有助于抗病毒药物的开发其作用方式是融合抑制的疗法也是病毒疫苗的目标。拟议的研究重点是血凝素 HA2 和 gp41 融合蛋白。分别选择流感病毒和人类免疫缺陷病毒的这些蛋白质是因为它们。病毒会引起主要的人类疾病，并且因为这些蛋白质是其他 I 类病毒的原型尽管蛋白质序列是非同源的，但这两种蛋白质都是单通道的。病毒膜的跨膜蛋白，具有约 180 个残基的胞外域区域，位于病毒膜之外病毒特别强调约 25 个残基的 N 端“融合肽”(FP) 结构域。在融合催化中发挥关键作用，并被认为在融合过程的早期插入宿主细胞膜。有四个目标，其中包括HA2（目标1）和gp41（目标2）的FP的膜中的结构，以及 HA2(Aim 3) 和 gp41(Aim 4) 的 FP 的膜位置分为 (a) 仅 FP 和 (b) FP+可溶性胞外域+跨膜结构域部分两种蛋白质的 FP 可以采用任一 α。螺旋或反平行的β折叠结构，这两种结构都催化膜融合。 α HA2 和 β gp41 FP 结构通过推论得到进一步发展。对 HA2 G1E 和 gp41 V2E 突变体的测量，这两种突变体都会导致融合和病毒严重受损阐明 FP 结构和膜接触的主要技术是固态核磁。共振（SSNMR），特别强调旋转回波双共振（REDOR），它可以阐明核间邻近度和距离 ≤10Å 将用于确定螺旋间 HA2 FP。 β gp41 FP 结构的结构和反平行登记将被确定。不同的结构以及结构和群体如何随着融合损害突变而变化。将进行 FP/膜接触的 REDOR 测量以及互补的 SSNMR 使用顺磁弛豫和 1H 自旋扩散测量 FP/水接触。 OMB 编号 0925-0001/0002（修订版 03/16 已批准至 10/31/2018）页面延续格式页面