The use of ESI-MS to probe the binding of divalent cations to calmodulin.
Proteins have evolved with distinct sites for binding particular metal ions. This allows metalloproteins to perform a myriad of specialized tasks with conformations tailor-made by the combination of its primary sequence and the effect on this of the ligated metal ion. Here we investigate the selectivity of the calcium trigger protein calmodulin for divalent metal ions. This ubiquitous and highly abundant protein exists in equilibrium between its apo and its holo form wherein four calcium ions are bound. Amongst its many functions, calmodulin modulates the calcium concentration present in cells, but this functional property renders it a target for competition from other metal ions. We study the competition posed by four other divalent cations for the calcium binding sites in calmodulin using electrospray ionization mass spectrometry (ESI-MS). We have chosen two other group II cations Mg(2+), Sr(2+), and two heavy metals Cd(2+), Pb(2+). The ease with which each of these metals binds to apo and to holo CaM[4Ca] is described. We find that each metal ion has different properties with respect to calmodulin binding and competition with calcium. The order of affinity for apo CaM is Ca(2+) >> Sr(2+) approximately Mg(2+) > Pb(2+) approximately Cd(2+). In the presence of calcium the affinity alters to Pb(2+) > Ca(2+) > Cd(2+) > Sr(2+) > Mg(2+). Once complexes have been formed between the metal ions and protein (CaM:[xM]) we investigate whether the structural change which must accompanies calcium ligation to allow target binding takes place for a given CaM:[xM] system. We use a 20 residue target peptide, which forms the CaM binding site within the enzyme neuronal nitric-oxide synthase. Our earlier work (Shirran et al. 2005) [1] has demonstrated the particular selectivity of this system for CaM:4Ca(2+). We find that along with Ca(2+) only Pb(2+) forms complexes of the form CaM:4M(2+):nNOS. This work demonstrates the affinity for calcium above all other metals, but also warns about the ability of lead to replace calcium with apparent ease.
Shirran
Sally L
SL
School of Chemistry, University of Edinburgh, Edinburgh, United Kingdom.
Barran
Perdita E
PE
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002104:Cadmium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism*; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D020035:Holoenzymes / Q000378:metabolism; D007854:Lead / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013324:Strontium / Q000378:metabolism
D001051:Apoenzymes; D002147:Calmodulin; D002413:Cations, Divalent; D020035:Holoenzymes; D011994:Recombinant Proteins; D002104:Cadmium; D007854:Lead; D052248:Nitric Oxide Synthase Type I; D008274:Magnesium; D002118:Calcium; D013324:Strontium