Chapter 4: Mutagenesis of the calmodulin binding domain of neuromodulin

This chapter focuses on biochemical properties of neuromodulin that may be important for its function in neurons. Two interesting biochemical properties that may be important for the physiological function of neuromodulin are its affinity for CaM and the effect of protein kinase C phosphorylation on CaM interactions. The concentration of neuromodulin in the brain and the affinity of the protein for CaM in the absence of free calcium are sufficient to complex the majority of CaM present. Consequently, interactions between neuromodulin and CaM as well as the regulation of CaM binding by protein kinase C phosphorylation are of considerable interest. The protein kinase C phosphorylation site of neuromodulin is serine-41. This phosphorylation significantly reduces the affinity of neuromodulin for CaM, suggesting that the introduction of negative charge may abolish CaM binding. The chapter addresses this issue by substituting serine-41 with an aspartate or an asparagines residue. The aspartate-41 mutant neuromodulin did not bind to CaM-Sepharose. In contrast, the asparagine-41 mutant bound to CaM-Sepharose in a manner indistinguishable from the wild type protein.