Mass Mapping of Macromolecular Assemblies

钙/钙调蛋白依赖的蛋白激酶II是一种 多功能丝氨酸/苏氨酸蛋白激酶广泛存在于 神经元。最近，有证据表明，海马区CaM 存在于突触后密度中的激酶II起着重要的作用。 在长期增强中。已知这种蛋白质会组装成一种 8到10个亚基的多聚体和由此产生的 全酶(MR=500 kDa)具有自身磷酸化能力 在钙/钙调蛋白启动后。扫描传输 电子显微镜(STEM)和电子能量损失谱 (鳗鱼)正被用来确定：质量在宇宙中的分布 细胞间黏附分子激酶II的结合和催化结构域 在存在的情况下与全酶结合的钙调蛋白 钙；以及细胞的磷酸化和钙含量 全酶。将样品放入液态乙烷中冷冻， 冷冻移植到STEM中并进行数字成像。量化 对所得质谱图的分析提供了分子量 全酶的分布。实验正在进行中，以 测量单个分子的磷酸化水平。这是一个 继续开展校内研究项目Z01-RR-10327-09 贝儿。

Ca2+/calmodulin-dependent protein kinase II is a multifunctional serine/ threonine protein kinase that is ubiquitous in neurons. Recently, there has been evidence that hippocampal CaM kinase II, present in post-synaptic densities, plays an important role in long term potentiation. The protein is known to assemble into a multimer of between 8 and 10 subunits and the resulting holoenzyme (Mr = 500 kDa) is capable of auto-phosphorylation after being primed by Ca/calmodulin. Scanning transmission electron microscopy (STEM) and electron energy loss spectroscopy (EELS) are being used to determine: the distribution of mass in the association and catalytic domains of CAM kinase II; the number of calmodulins that are bound to the holoenzyme in the presence of calcium; and the phosphorylation and calcium content of the holoenzyme. Specimens are plunge frozen into liquid ethane, cryotransferred into the STEM and digitally imaged. Quantitative analysis of the resulting mass maps provide molecular weight distributions of the holoenzyme. Experiments are in progress to measure the phosphorylation level of individual molecules. This is a continuation of Intramural Research Project Z01-RR-10327-09 BEI.