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Controlling the interaction between CaMKII and Calmodulin with a photocrosslinking unnatural amino acid

Iva Lučić,Pin-Lian Jiang,Andreas Franz,Yuval Bursztyn,Fan Liu,A. Plested

Published 2023 in Protein Science

ABSTRACT

Using unnatural amino acid mutagenesis, we made a mutant of CaMKII that forms a covalent linkage to Calmodulin upon illumination by UV light. Like wild‐type CaMKII, the L308BzF mutant stoichiometrically binds to Calmodulin, in a calcium‐dependent manner. Using this construct, we demonstrate that Calmodulin binding to CaMKII, even under these stochiometric conditions, does not perturb the CaMKII oligomeric state. Furthermore, we were able to achieve activation of CaMKII L308BzF by UV‐induced binding of Calmodulin, which, once established, is further insensitive to calcium depletion. In addition to the canonical auto‐inhibitory role of the regulatory segment, inter‐subunit crosslinking in the absence of CaM indicates that kinase domains and regulatory segments are substantially mobile in basal conditions. Characterization of CaMKIIL308BzF in vitro, and its expression in mammalian cells, suggests it could be a promising candidate for control of CaMKII activity in mammalian cells with light.

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