Learning and memory formation at the cellular level involves decoding complex electrochemical signals between nerve cells, or neurons. Understanding these processes at the molecular level requires a comprehensive study of calcium-sensitive proteins that serve as signal mediators within cells. More specifically, the protein calcium/calmodulin-dependent protein kinase II (CaMKII) is a key regulator of downstream cellular signaling events in the brain, playing an important role in long term memory formation. CaMKII is encoded in humans on four different genes: alpha, beta, gamma and delta. For added complexity, each of these gene products can be alternatively spliced and translated into multiple protein variants. Each protein forms an oligomer comprised of 12-14 subunits, and each subunit is made of a kinase domain, regulatory segment, variable linker region and hub domain. Through decades of animal studies, it is known that alpha and beta CaMKII proteins are most abundant in the brain. The initial activating signal of CaMKII is calcium-bound calmodulin (Ca2+/CaM) which binds to the regulatory segment. The first chapter of this dissertation introduces what is currently known of the relationship between CaMKII structure and activity regulation and the motivation to pursue the study of activation properties among splice variants and ancestrally resurrected CaMKII proteins. The second chapter reviews calcium signaling in the brain and reproductive system. The third chapter explores differences in Ca2+/CaM sensitivity of alpha and beta CaMKII splice variants, and reveals a previously unknown allosteric role for the hub domain. The results from this study led to a biochemical and biophysical characterization of ancestrally resurrected CaMKII splice variants to further explore the contributions from the hub domain and dissect how amino acid residue positions within this domain may be important in regulating kinase activity, as written in the fourth chapter. The fifth chapter summarizes some key takeaways I’ve learned from the time in the Stratton lab and gives insight for future students taking over the projects. The sixth and final chapter is an Appendix summarizing results of a pull-down assay used to study protein-protein interactions between human CaMKII alpha and beta hub domains and potential interactors found in mouse brain lysate.