ELECTRON MICROSCOPY OF MACROMOLECULAR STRUCTURES

大分子结构的电子显微镜

• 批准号: 3268608
• 负责人: HENRY S SLAYTER
• 金额: $ 12.15万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-04-01 至 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3268608
• 关键词: antibody formation; antibody specificity; blood proteins; chemical structure; complement; conformation; electron microscopy; fibrinogen; fibronectins; gel electrophoresis; gel filtration chromatography; glycoproteins; human tissue; immunoglobulins; laboratory mouse; laboratory rabbit; lyophilization; macromolecule; membrane activity; membrane proteins; mucins; muscle proteins; myosins; polymers; radioimmunoassay; radiotracer; tissue /cell preparation

The proposed research will improve methods for high-resolution EM of individual macromolecules, and will pursue applications of these to cogent biological problems. Coating by thin refractory metal films effectively contrasts individual macromolecular substructure. Due to recent improvements in electron optics, grain size, rather than instrumental factors, currently limits ultimate resolution. Thus, we intend to produce fine-grain coatings which will fully exploit available resolving power, particularly applying high-contrast darkfield imaging. We will explore use of mixtures of refractory metals (which should limit possibilities for crystallization and thus reduce crystallite size) and of low temperature specimen supports (which should favor film continuity). Effects of solvent systems and drying conditions will also be studied. These techniques will be applied to a number of macromolecular systems, including myosin, fibrinogen, epiglycanin, retinol-binding protein, antigen-antibody complexes, bronchial mucins, and hamster female protein. We plan applications of the electronmicroscopic mapping approach, in which macromolecules are complexed with readily-resolvable, site-specific protein markers, to a number of problems. Chemically-defined sites in the fibrinogen molecule, including the important region which binds factor XIII, staphylococcin and blood platelets, will be located by complexing with specifically-directed Fabs or with monoclonal antibodies. Positions of the 4 carbohydrate residues in this molecule will be determined by complexing with specific lectins. We will further characterize cardiac myosin in a combined physical-chemical and electron microscopic study, as well as assessing the effects of various physiological conditions on function and structure of myosin S1 region, and polymeric forms. Cardiac myosin will be mapped with specific Fabs directed against subregions of these molecules (S1, S2, light chains, S1 subdomains and hinge) in order to elucidate topography of the myosin head region. Positions of specific epitopes and carbohydrate residues along epiglycanin (a mouse mammary tumor cell-surface glycoprotein) will be mapped by a series of specific lectins, and by rabbit and monoclonal IgG and IgM. The latter will be used to determine the specificities of these antibodies for epiglycanin, and for the monospecific site, respectively. Certain other large glycoproteins will also be mapped.

提出的研究将改进高分辨率电磁成像的方法。 单个大分子，并将寻求这些应用于有说服力的 生物问题。 薄薄的难熔金属薄膜涂层有效地对比了个体 大分子亚结构。由于最近电子技术的进步 光学，颗粒大小，而不是仪器因素，目前限制 终极解决方案。因此，我们打算生产细晶涂层， 将充分利用可用的分辨率，特别是应用 高对比度暗场成像。我们将探索混合使用 难熔金属(这将限制结晶和 从而减小了微晶尺寸)和低温样品载体 (这应该有利于电影的连续性)。溶剂体系的影响和 还将研究干燥条件。这些技术将被应用于 一些大分子系统，包括肌球蛋白，纤维蛋白原， 表没食子儿茶素蛋白，视黄醇结合蛋白，抗原抗体复合物，支气管 粘蛋白和仓鼠雌性蛋白。 我们计划电子显微镜测绘方法的应用，其中 大分子与易分解的、特定于部位的蛋白质络合。 标记，以解决一些问题。化学定义的位置在 纤维蛋白原分子，包括结合因子的重要区域 XIII，葡萄球菌素和血小板，将通过络合定位 使用特定定向的Fbs或使用单抗。职位 该分子中的4个碳水化合物残留量将由 与特定的凝集素结合。我们将进一步描述心脏的特征 肌球蛋白在物理-化学和电子显微镜相结合的研究中，AS 以及评估各种生理条件对 肌球蛋白S1区的功能和结构，以及聚合体形式。心脏 肌球蛋白将与针对亚区的特定Fabs进行映射 这些分子(S1、S2、轻链、S1亚区和铰链)以 阐明肌球蛋白头部区域的地形图。特定位置的 表观糖蛋白(一种小鼠乳腺肿瘤)的表位和碳水化合物残基 细胞表面糖蛋白)将被一系列特定的凝集素映射， 兔和单抗的免疫球蛋白和免疫球蛋白。后者将被用来 确定这些抗体对表观甘露聚糖的特异性，以及对 分别为单特异性位点。某些其他大的糖蛋白 也将被映射。