INTERACTIONS BETWEEN DOMAINS OF APO CALMODULIN ALTER CALCIUM-BINDING AND STABILITY

Calmodulin (CaM) is an essential protein that exerts exquisite spatial and temporal control over diverse eukaryotic processes. Although the two half-molecule domains of CaM each have two EF-hands and bind two c alcium ions cooperatively, they have distinct roles in activation of s ome targets. Interdomain interactions may mediate coordination of thei r actions. Proteolytic footprinting titrations of CaM [Pedigo and Shea (1995) Biochemistry 34, 1179-1196; Shea, Verhoeven, and Pedigo (1996) Biochemistry 35, 2943-2957] showed that calcium binding to the high-a ffinity sites (III and IV in the C-domain) alters the conformation of helix B in the N-domain despite sites I and II being vacant, This may arise from calcium-induced disruption of interactions between the apo domains, In this study, comparing the cloned domains (residues 1-75, 7 6-148) to whole CaM, the proteolytic susceptibility of helix B in the apo isolated N-domain was higher than in apo CaM, The isolated N-domai n was monotonically protected by calcium binding and had a higher calc ium affinity than when part of whole CaM. The change in affinity was s mall (1-1.5 kcal/mol) but acted to separate the domain saturation curv es of whole CaM. Unfolding enthalpies and melting temperatures of the apo isolated domains did not correspond to the two transitions resolve d for apo CaM. In summary, the interactions between domains of apo CaM protected the N-domain from proteolysis and raised its T-m by 10 degr ees C, demonstrating that CaM is not the sum of its parts.