STUDIES OF CONFORMATIONAL CHANGES IN CA2+/CALMODULIN DEPENDENT KINASE II USING S

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal is for using SAXS to study the conformational changes that occur in calmodulin and its associated kinase upon calmodulin binding and trapping. Multifunctional Ca2+/calmodulin dependent kinase II (CaMKII) is an oligomeric, widely expressed serine/threonine kinase that plays a role in many cellular functions including cell cycle regulation, protein secretion, apoptosis, and gene expression. However, CaMKII has been most studied in relation to its role in synaptic plasticity, and particularly long-term potentiation. Here, abundant evidence has implicated CaMKII as one of the key molecules that regulate the strength of synapses. CaMKII is activated upon binding of Ca2+-bound calmodulin (CaM). If the level of stimulation is sufficiently strong, CaMKII undergoes autophosphorylation upon CaM binding. This alters the structural properties of the enzyme in an unknown fashion such that CaMKII now retains a high level of activity even upon removal of Ca2+. Furthermore, a second result of autophosphorylation is that the affinity of CaM for CaMKII is substantially increased due to a >1000-fold decrease in the CaMKII-CaM dissociation rate, a phenomenon referred to as CaM trapping. Due to autophosphorylation and/or CaM trapping, CaMKII now remains active for a sufficient period of time to initiate the downstream signaling processes that alter synaptic strength. In order to facilitate detection of the structural changes in CaMKII using SAXS, a monomeric form of the kinase will be employed. The data will initially be analyzed in the form of a Guinier plot to extract the radius of gyration of the particular particle under examination. The pair distribution function will also be examined to extract shape information about each particle. Subsequently, algorithms that facilitate construction of low-resolution molecular models will be employed to generate three dimensional density maps from the scattering data