Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca²⁺/calmodulin‐dependent kinase II

Abstract

Abstract Calmodulin (CaM) functions depend on interactions with CaM‐binding proteins, regulated by . Induced structural changes influence the affinity, kinetics, and specificities of the interactions. The dynamics of CaM interactions with neurogranin (Ng) and the CaM‐binding region of /calmodulin‐dependent kinase II (CaMKII 290−309 ) have been studied using biophysical methods. These proteins have opposite dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that and CaM interact very rapidly, and with moderate affinity ( ). Calmodulin‐CaMKII 290−309 interactions were only detected in the presence of , exhibiting fast kinetics and nanomolar affinity ( ). The CaM–Ng interaction had higher affinity under ‐depleted ( and k −1 = 1.6 × 10 −1 s −1 ) than ‐saturated conditions ( ). The IQ motif of Ng (Ng 27−50 ) had similar affinity for CaM as Ng under ‐saturated conditions ( ), but no interaction was seen under ‐depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng ( ) and CaMKII 290−309 ( ) interactions. Although CaMKII 290−309 showed expected interaction characteristics, they may be different for full‐length CaMKII. The data for full‐length Ng, but not Ng 27−50 , agree with the current model on Ng regulation of /CaM signaling.