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

• 批准号: 8209021
• 负责人: David P Weliky
• 金额: $ 32.84万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 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/8209021
• 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; public health relevance; receptor; research study; solid state nuclear magnetic resonance; vaccine development

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膜融合的基础，并了解原子分辨率细节。病毒和靶细胞膜的融合是许多在疾病中重要的病毒感染的关键步骤，对融合机制的详细理解将有助于开发抗病毒疗法，其作用方式是融合抑制。融合蛋白也是病毒疫苗发育的靶标。拟议的研究集中于各个人类免疫缺陷病毒和流感病毒的GP41和Hemagglutin fusion蛋白。选择这些蛋白质是因为病毒引起主要的人类疾病，并且因为蛋白质是其他I类病毒融合蛋白的原型。特别强调了融合蛋白的N末端“融合肽”（FP）结构域，该结构蛋白代表蛋白质N末端的约20个残留的极性区域。大量的生化和生物物理研究表明，FP在融合中起关键作用，并且具有FP序列的化学合成肽可以用作了解病毒/靶细胞融合的某些方面的有用模型系统。 长期目标将以三个特定的目标实现。 AIM 1专注于生产包含FPS（包括全长HA2）的GP41和HA2蛋白的大型GP41和HA2蛋白的生产。另外，将制造包含C末端膜透明外部区域（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，MPE也是HIV疫苗靶标。膜相关构建体的残留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）测量确定。将特别关注：（1）测试短肽研究中FP和MPER的结构模型是否对大型构造中的这些区域有效； （2）将FP和MPER结构与观察到的构造的融合性相关联； （3）检查GP41 FP是否接近MPER还是TM。 FP通过FP阻塞MPER可以降低MPER疗效作为免疫原。 AIM 2专注于确定这些大的GP41和HA2构建体中FP，MPER和TM区域的膜位置，使用蛋白13CO与脂质或胆固醇或胆固醇31P，19F或2H之间距离的SSNMR测量值。这些实验将测试深层而不是跨膜FP膜插入的一般结构 - 功能模型与更大的膜扰动和更快的融合催化相关。 AIM 3的重点是确定B纸FP的反平行登记册在几种具有非常不同的FP膜位置和融合性的GP41构建体中的分布。将估计FP膜插入的自由能，并与膜位置和融合性相关。总体目标是连接高分辨率FP结构，膜插入深度和融合性。 公共卫生相关性：拟议的研究将为人类免疫缺陷病毒和流感病毒进入宿主细胞以及如何停止该条目提供深入了解。此外，该研究将提供有关如何设计HIV疫苗的信息。