Calcium-Induced Structural Transitions of the Calmodulin-Melittin System Studied by Electrospray Mass Spectrometry: Conformational Subpopulations and Metal-Unsaturated Intermediates

Calmodulin (CaM) is a calcium-sensing protein that can bind to and activate various target enzymes Here, electrospray ionization mass spectrometry (ESI-MS) was used to investigate calcium-induced structural changes of CaM, as well as binding to the model target melittin (Mel). Nonspecific metalation artifacts were eliminated by conducting the experiments in negative ion mode and with calcium tartrate as metal soul cc [Pan et al. (2009) Anal. Chew 81, 5008] Two coexisting CaM subpopulations can be distinguished on the basis of their ESI charge state distributions, namely, relatively disordered conformers (CaMD, high charge states) and more tightly folded proteins (Ca M-I., low charge states) Calcium titration experiments on isolated CaM reveal that the transition from apo-CaMD to Ca-4 center dot CaMF proceeds with apparent K-d values of 10,14,30, and 12 mu M In the presence of Mel, a gradual [Ca2+] increase results in an overall population shift from apo-CaMD to Ca-4 center dot CaMF center dot Mel. This transition involves various intermediates, Ca-n center dot CaMF center dot Mel with n = 0, ..., 3, as well as apo-CaMD center dot Mel. Thus, neither the binding of four Ca2+ nor the existence of a tightly folded Ca M conformation is a prerequisite for target binding. Millisecond time-resolved ESI-MS experiments were conducted to monitor the response of a premixed CaM-Mel solution to a calcium concentration jump, thereby mimicking the conditions encountered in a cellular signaling context. The resulting data suggest that the formation of Ca-4 center dot CaMF center dot Mel proceeds along three parallel kinetic pathways: (1) metal binding to CaMD followed by formation of a compact protein-target complex, (2) folding of the apoprotein, then target binding, followed by metal complexation, (3) target binding to apo-CaMD followed by sequential metal binding. The exact structural properties of the various metal-unsaturated CaM species, as well as their physiological roles, remain to be elucidated.