Mass Mapping of Macromolecular Assemblies

Ca 2 +/钙调蛋白依赖性蛋白激酶II是一种 多功能丝氨酸/苏氨酸蛋白激酶是普遍存在于 神经元最近，有证据表明海马钙调素 存在于突触后致密物中的激酶II在突触后致密物中起重要作用， 在长时程增强中。已知这种蛋白质会组装成 8至10个亚基的多聚体， 全酶（Mr = 500 kDa）能够自磷酸化 经Ca/钙调素预处理后。扫描透射 电子显微镜（STEM）和电子能量损失谱 （EELS）用于确定： CAM激酶II的结合和催化结构域; 钙调素，在存在下与全酶结合 钙;和磷酸化和钙含量的 全酶将样本放入液态乙烷中冷冻， 冷冻转移到STEM中并进行数字成像。定量 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， 测量单个分子的磷酸化水平。这是一 校内研究项目Z 01-RR-10327-09的延续 贝

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.