Mass Mapping of Macromolecular Assemblies

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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.