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：
8050402
负责人：
David P Weliky
金额：
$ 29.36万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-02-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8050402
关键词：
Abbreviations Adopted Antigens Antiviral Agents Binding Biochemical Biological Models C-terminal Catalysis Cell Nucleus Cell fusion Cell membrane Cells Chemicals Chimeric Proteins Cholesterol DC-specific ICAM-3 grabbing nonintegrin Data Data Analyses Development Disease Electron Microscopy Epitopes Free Energy Freeze Fracturing Funding Goals Grant HIV HIV Envelope Protein gp120 HIV Fusion Inhibitors HIV Infections HIV vaccine Hemagglutinin Infection Label Left Length Lipids Location Measurement Membrane Membrane Fusion Modeling Molecular Conformation N-terminal Nuclear Magnetic Resonance Peptides Pharmaceutical Preparations Play Point Mutation Production Proteins Registries Research Resistance development Resolution Site Solid Structural Models Structure T-20 Tertiary Protein Structure Testing Therapeutic Transmembrane Domain Vertebral column Vesicle Viral Viral Fusion Proteins Viral Proteins Viral Vaccines Virion Virus base cost design human disease influenzavirus inhibitor/antagonist insight neutralizing antibody peptide structure protein aminoacid sequence prototype receptor research study solid state nuclear magnetic resonance vaccine development

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand the basis for viral fusion protein-induced membrane fusion with atomic-resolution detail. 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 gp41 and the hemagglutinin HA2 fusion proteins of the respective human immunodeficiency virus and influenza virus. These proteins are chosen because the viruses cause major human diseases and because the proteins serve as prototypes for other class I viral fusion proteins. There is particular emphasis on the N-terminal "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 three specific aims. Aim 1 focuses on production of multi-mg quantities of large constructs of gp41 and HA2 proteins that contain FPs, including full-length HA2. In addition, gp41 constructs will be made which contain the C-terminal membrane-proximal external region (MPER) and viral transmembrane (TM) domains. MPER and TM are important in fusion and MPER is also an HIV vaccine target. Residue-specific conformation of the membrane-associated constructs will be determined with solid- state nuclear magnetic resonance (SSNMR) measurements of specific carbonyl (13CO) chemical shifts. There will be particular focus on: (1) testing whether structural models of FP and MPER from short peptide studies are valid for these regions in the large constructs; (2) correlating FP and MPER structure to the observed fusogenicity of the construct; and (3) examining whether the gp41 FP is close to either the MPER or TM. Occlusion of MPER by FP could reduce MPER efficacy as an immunogen. Aim 2 focuses on determining the membrane locations of FP, MPER, and TM regions in these large gp41 and HA2 constructs using SSNMR measurements of distances between protein 13CO and lipid or cholesterol 31P, 19F, or 2H. These experiments will test the general structure-function model that deep but not transmembrane FP membrane insertion correlates with greater membrane perturbation and more rapid fusion catalysis. Aim 3 focuses on determining the distribution of antiparallel registries of b sheet FP in several gp41 constructs with very different FP membrane locations and fusogenicities. The free energies of FP membrane insertion will be estimated for the observed registries and correlated with membrane locations and fusogenicities. The overall goal is to connect high-resolution FP structure, membrane insertion depth, and fusogenicity. PUBLIC HEALTH RELEVANCE: The proposed research will provide insight into entry of human immunodeficiency virus and influenza virus into host cells and how this entry can be stopped. In addition, the research will provide information about how a HIV vaccine should be designed.

描述（由申请人提供）：拟议研究的长期目标是了解病毒融合蛋白诱导的具有原子分辨率细节的膜融合的基础。病毒和靶细胞膜的融合是许多疾病中重要病毒感染的关键步骤，对融合机制的详细了解将有助于开发其作用方式为融合抑制的抗病毒疗法。融合蛋白也是病毒疫苗开发的目标。拟议的研究重点是人类免疫缺陷病毒和流感病毒各自的 gp41 和血凝素 HA2 融合蛋白。选择这些蛋白质是因为这些病毒会引起主要的人类疾病，并且这些蛋白质可以作为其他 I 类病毒融合蛋白的原型。特别强调融合蛋白的 N 端“融合肽”(FP) 结构域，它代表蛋白 N 端约 20 个残基的非极性区域。大量的生化和生物物理学研究表明，FP 在融合中发挥着关键作用，并且具有 FP 序列的化学合成肽可以作为有用的模型系统来了解病毒/靶细胞融合的某些方面。长期目标将通过三个具体目标来实现。目标 1 侧重于生产多毫克数量的含有 FP（包括全长 HA2）的 gp41 和 HA2 蛋白大型构建体。此外，将制备包含C端近膜外部区域(MPER)和病毒跨膜(TM)结构域的gp41构建体。 MPER 和 TM 在融合中很重要，MPER 也是 HIV 疫苗的靶点。膜相关构建体的残基特异性构象将通过特定羰基（13CO）化学位移的固态核磁共振（SSNMR）测量来确定。将特别关注：（1）测试短肽研究中的 FP 和 MPER 结构模型对于大型构建体中的这些区域是否有效； (2) 将 FP 和 MPER 结构与观察到的构建体融合性相关联； (3)检查gp41 FP是否接近MPER或TM。 FP 阻断 MPER 可能会降低 MPER 作为免疫原的功效。目标 2 重点是使用 SSNMR 测量蛋白质 13CO 与脂质或胆固醇 31P、19F 或 2H 之间的距离来确定这些大型 gp41 和 HA2 构建体中 FP、MPER 和 TM 区域的膜位置。这些实验将测试一般的结构功能模型，即深度但非跨膜的 FP 膜插入与更大的膜扰动和更快速的融合催化相关。目标 3 侧重于确定 FP 膜位置和融合性差异很大的几种 gp41 构建体中 b 片 FP 反平行注册的分布。 FP 膜插入的自由能将根据观察到的记录进行估计，并与膜位置和融合性相关。总体目标是将高分辨率 FP 结构、膜插入深度和融合性联系起来。公共卫生相关性：拟议的研究将深入了解人类免疫缺陷病毒和流感病毒进入宿主细胞以及如何阻止这种进入。此外，该研究还将提供有关如何设计艾滋病毒疫苗的信息。