Monitoring changes in molecular conformation is essential for studying protein interactions within and between complexes. Current methods such as FRET or FCS can only reveal limited aspects of these changes, which in many cases is insufficient for interpretation. Fluorescent Polarization and Fluctuation Analysis (FPFA), a time-correlated single-photon counting technique that combines homo-FRET and FCS was developed to address this problem. In FPFA, changes in mass and/or shape of a protein complex, the number of fluorescent subunits per complex, as well as subunit proximity (1 - 10 nm), are simultaneously detected. Here we have extended FPFA by incorporating simultaneous hetero-FRET analysis so that additional structural changes can be monitored concurrently. This multi-modal approach was then used to observe changes in CaMKII holoenzyme in response to activation and subsequent interactions with NR2B. Revised FPFA revealed that catalytic-domain pairing is the fundamental structural motif of the holoenzyme organization. A genetically engineered dimeric-CaMKII functioned like native holoenzyme (8-14 subunits) in terms of activity, T286 autophosphorylation, structural changes triggered by calcium-calmodulin, and those associated with interaction with NR2B.