Multimodal microscopy can detect multiple changes in molecular conformation and has become a powerful tool for studying protein-protein interactions within and between complexes. Fluorescent Polarization and Fluctuation Analysis (FPFA), a time-correlated single-photon counting technique that combines homo-FRET, hetero-FRET and FCS was developed for this purpose. FPFA was recently extended by developing auto-FPFA, a robotic version of FPFA that can measure up to 96 biological samples automatically and/or repeatedly. Here we use auto-FPFA to investigate time-dependent changes of calcium/calmodulin-dependent protein kinase II (CaMKII) holoenzyme in response to activation and subsequent interaction with the T-site ligand motif of NR2B in vitro. Homo-FRET analysis showed that upon binding Ca2+/CaM, catalytic-domain pairing, a fundamental structural motif of the autoinhibited holoenzyme, rapidly disjoined, and then slowly reformed. The rate of this subsequent re-dimerization was influenced by ATP: the higher the ATP concentration, the slower the re-pairing rate. CaMKII binding to NR2B maintained the opening of catalytic-domain pairs, therefore suppressed the reformation of catalytic-domain pairing. Interestingly, hetero-FRET efficiency, an indicator of binding between mVenus-CaMKII and mCherry-NR2B, was proportional to the fraction of unpaired CaMKII subunits. These results suggest the existence of an ATP regulated time window for gating CaMKII interactions with T-site ligands.