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

计划总监/首席调查员（最后，第一，中间）：Weliky，David Paul 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的膜融合。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很重要，对融合机制的详细了解将有助于抗病毒的发展作用方式的治疗方法是融合抑制。融合蛋白也是病毒疫苗的靶标发展。拟议的研究侧重于血凝素HA2和GP41融合蛋白流感病毒和人类免疫缺陷病毒。选择这些蛋白质是因为这些病毒引起主要的人类疾病，并且因为蛋白质是其他I类病毒的原型融合蛋白。尽管蛋白质序列是非理论的，但两种蛋白都是单通蛋白病毒膜的跨膜蛋白，〜180个残留的外生域区域，位于外面病毒。特别强调了〜25个保留的N末端“融合肽”（FP）域，该结构域发挥了在融合过程中，在融合过程中插入宿主细胞膜中的关键作用。有四个目标包括HA2 FP（AIM 1）和GP41（AIM 2）的结构，以及 HA2 FP（AIM 3）和GP41（AIM 4）的FP的膜位置。每个目标都分为（a）仅FP，（b）FP+可溶性外生域+跨膜结构域的切片。两种蛋白质的FP都可以采用α 螺旋或反平行的β板结构，两种结构都催化膜融合。该项目的重点 αHA2和βGP41FP结构。结构/功能相关性通过推论进一步发展对HA2 G1E和GP41 V2E突变体的测量，这两者都会导致融合和病毒率极大受损感染。阐明FP结构和膜接触的主要技术是固态核磁共振（SSNMR）特别强调旋转回声双共振（重复）阐明核外接近率和距离≤10Å。重复将用于确定内螺旋ha2 fp βGP41FP结构的结构和反平行登记。将确定分数种群的不同的结构以及结构和种群如何随融合障碍突变而变化。 FP/膜触点以及完成的SSNMR将进行重新测量使用顺磁性松弛和1H自旋扩散对FP/水接触的测量。 OMB编号0925-0001/0002（Rev. 03/16批准通过10/31/2018）页面延续格式页面