The extent of conformational change that calcium binding induces in EF-hand proteins is a key biochemical property specifying Ca2+ sensor versus signal modulator function. To understand how differences in amino acid sequence lead to differences in the response to Ca2+ binding, comparative analyses of sequence and structures, combined with model building, were used to develop hypotheses about which amino acid residues control Ca2+-induced conformational changes. These results were used to generate a first design of calbindomodulin (CBM-1), a calbindin D-9k re-engineered with 15 mutations to respond to Ca2+ binding with a conformational change similar to that of calmodulin. The gene for CBM-1 was synthesized, and the protein was expressed and purified. Remarkably, this protein did not exhibit any non-native-like molten globule properties despite the large number of mutations and the nonconservative nature of some of them. Ca2+-induced changes in CD intensity and in the binding of the hydrophobic probe, ANS, implied that CBM-1 does undergo Ca2+ sensorlike conformational changes. The X-ray crystal structure of Ca2+-CBM-1 determined at 1.44 Angstrom resolution reveals the anticipated increase in hydrophobic surface area relative to the wild-type protein. A nascent calmodulin-like hydrophobic docking surface was also found, though it is occluded by the inter-EF-hand loop. The results from this first calbindomodulin design are discussed in terms of progress toward understanding the relationships between amino acid sequence, protein structure, and protein function for EF-hand CaBPs, as well as the additional mutations for the next CBM design.