posted on 2007-04-17, 00:00authored byCurt B. Boschek, Thomas C. Squier, Diana J. Bigelow
Binding of calcium to CaM exposes clefts in both N- and C-domains to promote their
cooperative association with a diverse array of target proteins, functioning to relay the calcium signal
regulating cellular metabolism. To clarify relationships between the calcium-dependent activation of
individual domains and interdomain structural transitions associated with productive binding to target
proteins, we have utilized three engineered CaM mutants that were covalently labeled with N-(1-pyrene)
maleimide at introduced cysteines in the C- and N-domains, i.e., T110C (PyC-CaM), T34C (PyN-CaM),
and T34C/T110C (Py2-CaM). These sites were designed to detect known conformers of CaM such that
upon association with classical CaM-binding sequences, the pyrenes in Py2-CaM are brought close together,
resulting in excimer formation. Complementary measurements of calcium-dependent enhancements of
monomer fluorescence of PyC-CaM and PyN-CaM permit a determination of the calcium-dependent
activation of individual domains and indicate the sequential calcium occupancy of the C- and N-terminal
domains, with full saturation at 7.0 and 300 μM calcium, respectively. Substantial amounts of excimer
formation are observed for apo-CaM prior to peptide association, indicating that interdomain interactions
occur in solution. Calcium binding results in a large and highly cooperative reduction in the level of
excimer formation; its calcium dependence coincides with the occupancy of C-terminal sites. These results
indicate that interdomain interactions between the opposing domains of CaM occur in solution and that
the occupancy of C-terminal calcium binding sites is necessary for the structural coupling between the
opposing domains associated with the stabilization of the interdomain linker to enhance target protein
binding.