113Cd NMR in the study of calcium binding proteins: Troponin C

Abstract

Calcium is one of nature’s most versatile metal ions, for example some 70 calcium binding proteins have been described [ 11. However there exists a marked lack of physical probes to study the properties of Ca’+ in these molecules in solution. Ca2’ is diamag- netic and has no convenient optic spectroscopic properties. The magnetic isotope 43Ca(1 = S/2; natural abundance 0.145%) may in principle be studied by NMR methods but direct observation of 4sCa NMR signals from calcium binding proteins is most probably ruled out by the large nuclear electric quadrupole moment of 43Ca. Due to very efficient quadrupole relaxation the NMR signal is expected to be broadened beyond detectability in binding sites with less than cubic symmetry [2,3]. The indirect NMR method, i.e. the study of NMR signals from small ions in solu- tion that undergo reasonably fast chemical exchange with macromolecular binding sites, has proved very informative for ions such as Nat and Cl- [4]. How- ever it is not expected to be generally applicable for the study of Ca2* binding proteins since if the calcium binding constant exceeds lo4 M-’ the chemical exchange rate of Ca” between protein and solvent is expected to be too slow at room temperature. ‘laCd NMR has in recent years emerged as a useful tool for the study of zinc proteins [S-9]. ‘13Cd has a spin I = l/2 magnetic nucleus and Cd’+ can often substitute for Zn2+ in zinc enzymes with at least partial retention of biological activity [lo]. The ionic radius