Biological functions for a large class of calmodulin-related proteins, such
as target protein activation and Ca2+ buffering, are based on fine-tuned b
inding and release of Ca2+ ions by pairs of coupled EF-hand metal binding s
ites. These are abundantly filled with acidic residues of so far unknown io
nization characteristics, but assumed to be essential for protein function
in their ionized forms. Here we describe the measurement and modeling of pK
(a) values for all aspartic and glutamic acid residues in apo calbindin D-9
k, a representative of calmodulin-related proteins. We point out that while
all the acidic residues are ionized predominantly at neutral pH, the onset
of proton uptake by Ca2+ ligands with high pK(a) under these conditions ma
y have functional implications. We also show that the negative electrostati
c potential is focused at the bidental Ca2+ ligand of each site, and that t
he potential is significantly more negative at the N-terminal binding site.
(C) 2001 Wiley-Liss, Inc.