Binary-FRET reveals transient excited-state structure associated with activity-dependent CaMKII - NR2B binding and adaptation

• Published in: Nature communications 2022-10, Vol.13 (1), p.6335-6335, Article 6335
• Main Authors: Nguyen, Tuan A., Puhl, Henry L., Hines, Kirk, Liput, Daniel J., Vogel, Steven S.
• Format: Article
• Language: English
• Subjects: 14; 14/33; 14/35; 14/69; 631/1647/2204; 631/378/2591; 631/57/2267; 631/57/2272; 96/33; 96/35; Adaptation; Affinity; Anisotropy; Binding; Ca2+/calmodulin-dependent protein kinase II; Calcium; Calcium (intracellular); Calcium - metabolism; Calcium signalling; Calcium-binding protein; Calcium-Calmodulin-Dependent Protein Kinase Type 2 - metabolism; Calmodulin; Dynamics; Energy transfer; Excitation; Fluorescence; Fluorescence Resonance Energy Transfer; Glutamic acid receptors; Holoenzymes - metabolism; Humanities and Social Sciences; Kinases; Ligands; multidisciplinary; Multiplexing; N-Methyl-D-aspartic acid receptors; Phosphorylation; Protein Binding; Protein interaction; Proteins; Receptors, N-Methyl-D-Aspartate - metabolism; Resonance; Science; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-33795-8

Synaptic functions are mediated and modulated by a coordinated choreography of protein conformational changes and interactions in response to intracellular calcium dynamics. Time-lapse 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. 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, at non-saturating free calcium concentrations, our multiplexed approach reveals that the holoenzyme exhibits a biochemical form of plasticity, calcium dependent adaptation of T-site ligand binding affinity. FRET can be used to study conformational changes and protein-protein interactions. Here the authors report Binary-FRET for monitoring two FRET reactions, one encoded in the fluorescence lifetime of the donor, another encoded in its anisotropy, and monitor the dynamics of CaMKII and its interaction with NR2B.