Mass Mapping of Macromolecular Assemblies

大分子组装体的质量作图

• 批准号: 6548617
• 负责人: Richard D Leapman
• 金额: --
• 依托单位: OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH
• 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/6548617
• 关键词: calmodulin dependent protein kinase; molecular weight; nerve /myelin protein; protein structure; scanning transmission electron microscopy; synapses

Scanning transmission electron microscopy (STEM) provides a versatile method for determining the molecular mass and hence the arrangement of subunits in large protein assemblies. Macromolecules are adsorbed onto a thin support film and a nanometer-sized electron probe is scanned across the specimen while the elastic-scattering signal is collected. The resulting digital image intensity is proportional to the local mass density of the specimen. Images can be recorded at low electron dose without significant radiation damage to the structures of interest. We have used this approach to characterize the composition of post-synaptic densities (PSDs) that are isolated from rat forebrain, frozen rapidly, and supported on thin carbon substrates. The PSD is a complex molecular machine, which underlies the post-synaptic membrane and is responsible for organizing receptors and signaling molecules at the post-synaptic active zone. It is found that PSDs become thicker upon exposure of the neurons to ischemia as well as other excitatory conditions and it is known that the enzyme CaMKII contributes to this thickening. We have used STEM mass mapping to estimate the number of CaMKII molecules that are added to the PSD under excitatory conditions. Knowledge of the total mass of the PSD opens the way to a quantitative and stochiometric analysis of functional changes in the protein composition of PSDs

扫描透射电子显微镜（STEM）提供了一种多功能方法，用于确定分子质量，从而在大蛋白质组件中的亚基排列。大分子被吸附到薄的支撑膜上，并在收集弹性散射信号的同时扫描了纳米尺寸的电子探针。所得的数字图像强度与试样的局部质量密度成正比。图像可以在低电子剂量下记录，而不会对感兴趣的结构进行重大辐射损害。我们已经使用了这种方法来表征从大鼠前脑分离出来的突触后密度（PSD）的组成，迅速冷冻并在薄碳底物上支撑。 PSD是一款复杂的分子机，它是后突触后膜的基础，并负责在后突触后活性区组织受体和信号分子。发现PSD在神经元暴露于缺血以及其他兴奋性条件下变得更厚，并且众所周知，CAMKII酶有助于这种增厚。我们已经使用了茎质量映射来估计在兴奋性条件下添加到PSD中的CAMKII分子的数量。了解PSD的总质量为PSDS蛋白质组成的功能变化的定量和静态分析开辟了道路