Mass Mapping of Macromolecular Assemblies

大分子组装体的质量作图

• 批准号: 8743765
• 负责人: Richard Leapman
• 金额: $ 9.35万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 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/8743765
• 关键词: Acceleration; Alzheimer' s Disease; Amino Acids; Amyloid Fibrils; Amyloid beta-Protein; Amyloidosis; Arctic Regions; Area; Bacteria; Biological; Cells; Characteristics; Circular Dichroism; Classification; Diffuse; Dimensions; Disease; Electron Microscope; Electron Microscopy; Electrons; Equipment and supply inventories; Exhibits; Genetic Polymorphism; Genome; Goals; Guns; Kinetics; Laboratories; Length; Life; Maps; Measurement; Measures; Membrane; Methods; Molecular; Morphology; Motor; Mutation; Pathway interactions; Peptides; Process; Proteins; Sampling; Scanning; Scanning Transmission Electron Microscopy Procedures; Shapes; Source; Spiroplasma; Structure; Techniques; Time; Toxic effect; beta pleated sheet; detector; fibrillogenesis; instrument; interest; macromolecular assembly; molecular mass; mutant; neutrophil; phospholipid inhibitor; protein distribution; rapid growth; research study; statistics; structural biology; transmission process; voltage

The scanning transmission electron microscope (STEM) has made significant contributions to structural biology by providing accurate determinations of the molecular masses of large protein assemblies that have arbitrary shapes and sizes. Nevertheless, STEM mass mapping has been implemented in very few laboratories, most of which have employed cold-field emission gun (FEG) electron sources operating at acceleration voltages of 100 kV and lower. Here we show that a 300 kV commercial transmission electron microscope (TEM) equipped with a thermally assisted Shottky FEG can provide accurate STEM mass measurements of disease-related amyloid fibrils. Morphology of aggregation intermediates and polymorphism of amyloid fibrils as well as aggregation kinetics of the Arctic mutant of the 40-amino acid Alzheimer's amyloid beta peptide (E22G), were explored using electron microscopy together with atomic force and circular dichroism measurements, which enabled the kinetics of aggregate formation to be correlated with the secondary structure of amyloid beta peptide. Scanning transmission electron microscopy (STEM) was employed to explore the fibril morphology and to measure mass-per-length of different fibril polymorphs. Characteristic dimensions of both intermediate aggregates and fibrils were measured during the aggregation process. Critical concentrations of fibrillization of both arctic mutation and wild type peptide were studied. At the end of a lag-period with almost indistinguishable changes in morphology of diffuse aggregates, the arctic mutation peptide underwent an abrupt transition to compact spherical aggregates with a highly distinct and homogeneous morphology. Aggregation then proceeded with a rapid growth of amyloid fibrils to produce a variety of morphologies, while the spherical aggregates eventually disappeared. It was found that amyloid fibrils exhibited a variety of polymorphs. At least four fibril polymorphs were identified by TEM and STEM and their mass-per-length statistics suggested supramolecular structures with two, four and six beta-sheet laminae. Real time aggregation dynamics of the fibrils revealed that different fibril morphologies not only co-exist in the same sample, but they also grow or dissociate at different rates. These results suggest an alternative pathway of fibrillogenesis for full-length Alzheimer's peptides with small and structurally ordered transient spherical aggregates as immediate precursors of amyloid fibrils. These findings could be significant in studying aggregation kinetics, morphology and interaction of peptide with inhibitors and phospholipid membranes of several other mutations of the Alzheimer's beta amyloid peptide. To elucidate how the components of an extremely simple cell are spatially organized, STEM mass mapping has been applied to analyze the molecular inventory of Spiroplasma melliferum, a wall-less free-living bacterium with an exceedingly small genome and dynamic helical geometry. Together with other biophysical measurements, STEM determination of the mass-per-length of whole Spiroplasma, the mass-per-length of the cell's cytoskelatal motor assemblies, and the mass-per-area of its membrane fractions, provide a general framework for a minimal inventory and arrangement of major cellular components that are needed for a minimal viable cell.

扫描透射电子显微镜（STEM）通过提供具有任意形状和大小的大型蛋白质组装体的分子质量的精确测定，对结构生物学做出了重大贡献。 尽管如此，STEM质量映射已经在很少的实验室中实施，其中大多数采用冷场发射枪（FEG）电子源，加速电压为100 kV或更低。在这里，我们表明，一个300千伏的商业透射电子显微镜（TEM）配备了热辅助的ShoeumFEG可以提供准确的干质量测量疾病相关的淀粉样纤维。 使用电子显微镜结合原子力和圆二色性测量，探索了淀粉样蛋白纤维聚集中间体和多态性的形态以及40个氨基酸的阿尔茨海默氏淀粉样蛋白β肽（E22 G）的北极突变体的聚集动力学，这使得聚集体形成的动力学与淀粉样蛋白β肽的二级结构相关。 采用扫描透射电子显微镜（STEM）来探索原纤维形态，并测量不同原纤维多晶型物的单位长度质量。 在聚集过程中测量中间聚集体和原纤维的特征尺寸。 研究了北极突变肽和野生型肽的絮凝临界浓度。在一个滞后期结束时，几乎难以区分的变化，在形态的弥漫性聚集体，北极突变肽经历了一个突然的过渡到紧凑的球形聚集体具有高度独特的和均匀的形态。聚集，然后进行与淀粉样纤维的快速增长，产生各种形态，而球形聚集体最终消失。结果发现，淀粉样纤维表现出多种多晶型。至少有四个原纤维多晶型物被确定为TEM和STEM和他们的质量每长度的统计表明超分子结构与两个，四个和六个β-片层。 原纤维的真实的时间聚集动力学揭示了不同的原纤维形态不仅共存于同一样品中，但它们也以不同的速率生长或解离。这些结果表明，全长阿尔茨海默氏肽的原纤维形成的替代途径，具有小的和结构有序的瞬时球形聚集体作为淀粉样蛋白原纤维的直接前体。这些发现在研究聚集动力学、形态学和肽与阿尔茨海默氏症β淀粉样肽的几种其他突变的抑制剂和磷脂膜的相互作用方面可能是重要的。 为了阐明一个非常简单的细胞的组成部分是如何在空间上组织的，STEM质量映射已被应用于分析螺原体melliferum，一个无壁的自由生活的细菌与一个非常小的基因组和动态螺旋几何的分子库存。 与其他生物物理测量一起，STEM测定整个螺原体的单位长度质量、细胞的细胞外马达组件的单位长度质量和其膜组分的单位面积质量，为最小活细胞所需的主要细胞组分的最小库存和安排提供了一个总体框架。