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.

扫描透射式电子显微镜(STEM)为结构生物学做出了重大贡献，它可以精确地测定具有任意形状和大小的大型蛋白质组件的分子质量。尽管如此，极少数实验室已经实施了STEM质量映射，其中大多数实验室使用了工作在100千伏及以下的加速电压下的冷场发射枪(FEG)电子源。在这里，我们展示了配备热辅助Shottky FEG的300千伏商用透射电子显微镜(TEM)可以提供疾病相关淀粉样纤维的准确STEM质量测量。 用电子显微镜结合原子力和圆二色谱研究了40个氨基酸的阿尔茨海默病淀粉样β蛋白(E22G)北极突变体的聚集中间体形态、淀粉样纤维的多态以及聚集动力学，使聚集形成的动力学与淀粉样β蛋白的二级结构相关联。用扫描电子显微镜(STEM)研究了不同晶型纤维的形态，并测量了不同晶型纤维的单位长度质量。在凝聚过程中测量了中间聚集体和纤维的特征尺寸。研究了北极突变多肽和野生型多肽的纤化临界浓度。在扩散聚集体的形态变化几乎不可区分的滞后期结束时，北极突变肽经历了一个突然转变为致密的球形聚集体，具有高度不同和均匀的形态。随着淀粉样蛋白纤维的快速生长，聚集产生了各种形态，而球形聚集最终消失了。研究发现，淀粉样蛋白纤维表现出多种多态。经透射电子显微镜和扫描电子显微镜鉴定，至少有四种纤维多晶型，其单位长度质量统计表明，超分子结构具有两个、四个和六个β-折叠片层。纤维的实时聚集动力学表明，不同的纤维形态不仅在同一样品中共存，而且它们以不同的速度生长或解离。这些结果表明，全长阿尔茨海默病多肽的另一种纤维形成途径是小的和结构有序的瞬时球形聚集体作为淀粉样蛋白纤维的直接前体。这些发现对研究阿尔茨海默病β淀粉样肽的聚集动力学、形态以及与其他几种突变的抑制剂和磷脂膜的相互作用具有重要意义。 为了阐明一个极其简单的细胞的成分是如何在空间上组织的，STEM质量图谱被应用于分析意大利螺旋体的分子清单，这是一种具有极小基因组和动态螺旋几何形状的无壁自由生活细菌。与其他生物物理测量一起，STEM测定整个螺旋体的每长度质量、细胞胞裂马达组件的每长度质量以及其膜组分的每面积质量，为最小限度地清点和安排最小存活细胞所需的主要细胞成分提供了一个总体框架。