Calcium signaling is a major regulatory system in cells and a crucial part of cell biology. An
important element in the decoding of intracellular calcium concentration into downstream
processes is the ubiquitous and highly conserved calcium binding protein calmodulin (CaM)
which can bind to and modulate the function of hundreds of different target proteins,
regulating such processes as synaptic plasticity, gene expression and electrical signaling. The
biophysical characterization of binding affinity and cooperative interactions between each of
calmodulin’s four EF-hand calcium binding sites is essential for understanding calcium
signaling. Highly conserved amino acid sequence differences in the ion binding loops of the
EF-hands give each site unique affinity for calcium. EF-hands are almost always found in
pairs, where binding to one of the sites affects the affinity of the paired site. We have used
spectroscopy to measure site-specific binding in each of the paired binding sites in the CaM
N-lobe, along with site-directed mutagenesis, to study the contributions of individual amino
acids to the ion binding affinity in the mutated site (cis effects) and in the neighboring site
(trans effects). Of the twelve amino acids in the binding loops, five are different between Site
1 and Site 2. We constructed proteins with substituted individual residues from Site 1 to Site
2. CaM with the full Site 1 sequence in both Site 1 and Site 2 shows significant changes in
affinity and binding characteristics in both sites. To investigate the contributions of the
individual amino acid differences, we made intermediate mutants containing individual amino
acid changes in Site 2. The cis-effects of the intermediate mutations on the mutated site, Site
2, seem to be independent and additive, whereas the trans-effects on the non-mutated Site 1
showed unexpected dependence on combinations of amino acid changes in Site 2.Neuroscienc
