Using multiphoton lifetime and anisotropy imaging to simultaneous monitor CaMKII activation, NR2B binding and holoenzyme translocation

Synaptic functions are mediated and modulated by protein conformational changes and interactions in response to intracellular calcium dynamics. Förster resonance energy transfer can be used to study the dynamics of both conformational changes and protein-protein interactions simultaneously under physiological conditions if two resonance energy transfer reactions can be multiplexed. Binary-FRET is a technique developed to independently monitor the dynamics of calcium calmodulin dependent protein kinase-II catalytic-domain pair separation in the holoenzyme, and its role in establishing activity-dependent holoenzyme affinity for the NR2B binding fragment of the N-methyl-D-aspartate receptor. When Binary-FRET is performed in an imaging mode the translocation of CaMKII can also be monitored as the kinase moves from the cytoplasm to the plasma membrane. Here we show that a transient excited-state intermediate exists where paired catalytic-domains in the holoenzyme first separate prior to subsequent NR2B association. Additionally, we show that translocation requires at least two functional binding sites for NR2B.