Structure And Assembly Of The Hepatitis B Nucleocapsid Protein

乙型肝炎核衣壳蛋白的结构和组装

• 批准号: 7964902
• 负责人: PAUL T WINGFIELD
• 金额: $ 71.66万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964902
• 关键词: Address; Affinity; Antibodies; Antigen-Antibody Complex; Antigens; B-Lymphocytes; Binding; Biotechnology; C-terminal; Cancer Etiology; Capsid; Capsid Proteins; Childhood; Chronic Hepatitis B; Clinical; Code; Collaborations; Complex; Cryoelectron Microscopy; Cytoplasm; Detection; Development; Diagnostic; Drug Delivery Systems; Elasticity; Encapsulated; Epitopes; Fab Immunoglobulins; Genes; Hepatitis B; Hepatitis B Core Antigen; Hepatitis B Virus; Hepatitis B e Antigens; Heterogeneity; Human; Image Analysis; Intervention; Kinetics; Knowledge; Label; Libraries; Life Cycle Stages; Maps; Mass Spectrum Analysis; Measurement; Measures; Metabolic; Methods; Modeling; Molecular; Molecular Structure; Monoclonal Antibodies; Mus; N-terminal; Nanotechnology; Nucleic Acids; Nucleocapsid; Nucleocapsid Proteins; Oryctolagus cuniculus; Particulate; Play; Population; Positioning Attribute; Process; Property; Protamines; Protein Precursors; Proteins; RNA-Directed DNA Polymerase; Reagent; Role; Structure; Surface; Surface Immunoglobulins; Surface Plasmon Resonance; System; Time; Universities; Vaccines; Viral Proteins; Virus; Work; X-Ray Crystallography; anti-hepatitis B; clinical Diagnosis; hepatitis B virus P protein; human disease; ion mobility; molecular assembly/self assembly; prevent; protein S precursor; serological marker; stoichiometry; tool; virus core

HBcAg has been expressed in E.coli were it assembles in the bacterial cytoplasm into icosahedral capsids, which contain bound host nucleic acid. Deletion of the polybasic C-terminal 34 residues (protamine domain) also produces assembly competent protein. The capsids from C-terminal truncated protein (Cp149) do not contain nucleic acid and their structure has been previously determined by cryo-electron microscopy and image analysis and by X-ray crystallography. Native HBeAg is also C-terminally truncated at position 149 and in addition contains a 10 residue N-terminal extension derived from partial processing of precursor protein (pre-C). Although detailed knowledge of the function and structure of HBeAg are unknown it has clinical importance as a serological marker. Using surface plasmon resonance (Biacore) to measure antibody antigen interactions, a kinetic-affinity map of a panel of monoclonal antibodies (mAbs) against HBV nucleocapsid proteins was determined. Monoclonal antibodies (murine origin) binding to the assembled (HBcAg) and non-assembled forms of the capsids (HBeAg) were identified and new combinations useful for clinical diagnosis described. Previous structural determinations of nucleocapsid-antibody immune complexes by cryo-electron microscopy helped to more clearly explain the immunological distinction between the assembled HBcAg and unassembled HBeAg antigen. This work has been extended to included human antibodies and the preliminary results indicate binding to distinct regions on the capsid surface (epitopes) which we had previously identified using the model murine antibodies. This work, together our previous description of an immune complex of capsids and an antibody representative of the surface immunoglobulin from naive B-cells, provide one of the most complete structural pictures of the interaction of antibodies with a viral protein system related to an important human disease. Using a rabbit antibody library, monoclonal antibodies (mAbs) were selected then humanized to produce chimeric mAb fragment antigen binding portions (Fab). Fabs against the HBV capsid proteins were selected for high affinity binding to the capsid subunits. The binding of the antibodies prevent the assembly of the capsids (a central functional and structural component of the HBV virus) and may with development provide useful anti-HBV reagents. Direct determination of the biophysical properties of the HBV capsid protein is limited due to the size of the multiprotein megadalton complex. In collaboration with Albert Heck (Utrecht University) macromolecular tandem and ion mobility mass spectrometry was used to study the stability and conformational diversity of HBV capsids. Very precise mass measurements were made and the exact molecular stoichiometries of HBV nucleocapsid complexes determined for the first time. This work was extended to include measurements of stability and elasticity of the capsids which resulted in the detection of conformational heterogeneity in the capsid population. Also, the stability of the capsids was addressed by the direct determination of the slow exchange of constituent subunits using isotopically labeled protein. The methods and approaches used have general applicability to the study of large molecular assemblies used in nano- and biotechnology

HBCAG已在大肠杆菌中表达，如果它在细菌细胞质中组装成含有结合的宿主核酸的二十面体c骨。多重基质C末端34残基（精完全结构域）的缺失也会产生组装胜任蛋白。来自C末端截短蛋白（CP149）的衣壳不含核酸，其结构先前已通过冷冻电子显微镜和图像分析以及X射线晶体学确定。天然HBEAG在149的位置也被C末端截断，此外，还包含10个残基N末端延伸，这些末端延伸源自前体蛋白的部分处理（PREC-C）。尽管对HBEAG的功能和结构的详细知识尚不清楚，但它作为血清学标记具有临床意义。使用表面等离子体共振（BIACORE）测量抗体抗原相互作用，确定了针对HBV Nucleocapsid蛋白的单克隆抗体（MABS）的动力学亲和力图。鉴定了与组合的（HBCAG）和不组装形式的衣壳（HBEAG）结合的单克隆抗体（鼠起源）（HBCAG）（HBEAG），并对所描述的临床诊断有用。通过冷冻电子显微镜对Nucleocapsid-抗体免疫复合物的先前结构测定有助于更清楚地解释组装的HBCAG和未组装的HBEAG抗原之间的免疫学区别。这项工作已扩展到包括人类抗体，初步结果表明与我们先前使用模型鼠抗体鉴定的衣壳表面（表位）上的不同区域结合。这项工作，我们以前对胶囊免疫复合物和来自天真B细胞表面免疫球蛋白的抗体的描述提供了抗体相互作用与与重要人类疾病有关的病毒蛋白系统相互作用的最完整结构图片之一。 使用兔抗体文库，选择人性化的单克隆抗体（mAb）以产生嵌合MAB片段抗原结合部分（FAB）。选择针对HBV衣壳蛋白的FAB以高亲和力与衣壳亚基的高亲和力结合。抗体的结合阻止了衣壳的组装（HBV病毒的中心功能和结构成分），并且可能随发育提供有用的抗HBV试剂。 由于多蛋白质Megadalton络合物的大小，HBV衣壳蛋白的生物物理特性的直接测定受到限制。与Albert Heck（Utrecht University）合作，使用了大分子串联和离子迁移率质谱法来研究HBV Capsids的稳定性和构象多样性。进行了非常精确的质量测量，并首次确定了HBV核素复合物的精确分子化学计量。这项工作扩展到包括测量帽的稳定性和弹性的测量，从而导致帽子种群中构象异质性的检测。同样，通过使用同位素标记的蛋白质直接确定成分亚基的慢速交换来解决衣壳的稳定性。所使用的方法和方法一般适用于纳米和生物技术中使用的大分子组件的研究