Chemistry Publications

Title

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

Document Type

Article

Publication Date

4-27-2010

Journal

Biochemistry

Volume

49

Issue

16

First Page

3477

Last Page

3486

URL with Digital Object Identifier

http://dx.doi.org/10.1021/bi100261c

Abstract

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 source [Pan et al. (2009) Anal. Chem. 81, 5008]. Two coexisting CaM subpopulations can be distinguished on the basis of their ESI charge state distributions, namely, relatively disordered conformers (CaM(D), high charge states) and more tightly folded proteins (CaM(F), low charge states). Calcium titration experiments on isolated CaM reveal that the transition from apo-CaM(D) to Ca(4).CaM(F) proceeds with apparent K(d) values of 10, 14, 30, and 12 microM. In the presence of Mel, a gradual [Ca(2+)] increase results in an overall population shift from apo-CaM(D) to Ca(4).CaM(F).Mel. This transition involves various intermediates, Ca(n).CaM(F).Mel with n = 0, ..., 3, as well as apo-CaM(D).Mel. Thus, neither the binding of four Ca(2+) nor the existence of a tightly folded CaM 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).CaM(F).Mel proceeds along three parallel kinetic pathways: (1) metal binding to CaM(D) 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-CaM(D) 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.