| Numerous cellular processes are regulated by changes in the intracellular [Ca2+]. Calmodulin (CaM) is the primary mediator of the Ca 2+-signal, transducing it into a variety of cellular responses. Since CaM is the main effecter of cellular response to the Ca2+-signal, characterization of the effects of target interaction and the intrinsic structural influences on CaM's Ca2+-binding properties is of great value to furthering the understanding of how a cell responds to a transient Ca 2+ flux.; It was determined that increasing the number of acid-pairs in the N-terminal Ca2+-binding sites of CaM increased the Ca2+ affinity. The maximum increases in Ca2+ affinity for site I require acid-pairs at any axis position (X, Y or Z), while maximal increases in Ca2+ affinity for site II required a Z-axis acid-pair. Additionally, completely filling the acid-pairs within the N-terminal Ca2+-binding sites of CaM is detrimental to high affinity Ca2+-binding. Furthermore, the increases in Ca2+ affinities with increasing number of acid-pairs are primarily due to an increase in the ion association rates for the Y and Z-axis acid-pairs, and a decrease in the Ca2+ dissociation rates for the X-axis acid-pair.; Additionally, it was determined that increasing the polarity within the N-terminal hydrophobic pocket of CaM increased Ca2+ affinity primarily due to decreasing the Ca2+ dissociation rate, while increasing the hydrophobicity demonstrated converse effects, implicating an inverse relationship between the polarity of hydrophobic pockets of CaM and the rates of Ca2+ dissociation. However, the effects of the glutamine substitutions within the hydrophobic pocket of CaM decreased Ca 2+ affinity without altering the Ca2+dissociation rate in the presence of the calmodulin binding peptide from calmodulin kinase II, while the hydrophobic substitutions within the hydrophobic pocket of CaM enhanced the Ca2+ binding properties of CaM in the presence of the calmodulin binding peptide from calmodulin kinase II. These data implicate the methionine residues within the Ca2+ dependently exposed hydrophobic surfaces as having an additional role in modulation of the Ca2+ binding properties of CaM by imparting an enhanced stability to the open and complexed states of CaM while not inhibiting the stability of any conformational state of CaM. |
