Lead-207 NMR: A novel probe for the study of calcium-binding proteins

The high-affinity Ca2+-binding sites of carp (pI 4.25) and pike (pI 5.0) parvalbumins, as well as those of mammalian calmodulin (CaM) and its C-terminal tryptic half-molecule (TR(2)C), were analyzed by Pb-207 NMR spectroscopy. For the parvalbumins, two Pb-207 signals were observed ranging in chemical shift from approximate to 750 to approximate to 1260 ppm downfield of aqueous Pb(NO3)(2), corresponding to Pb-207(2+) bound to the two high-affinity helix-loop-helix Ca2+-binding sites in each of these proteins. Four Pb-207 signals, which fall in the same chemical shift window, could be discerned for CaM. Experiments on TR(2)C permitted the assignment of each signal as due to Pb-207(2+) Occupying a helix-loop-helix site in either the N- or the C-lobe of the intact protein. Pb-207 and H-1 NMR titration studies on CaM provided evidence that Pb2+ binding to all four sites occurs simultaneously, in contrast to the behavior of this protein in the presence of Ca2+. Titrations of the Pb-207(2+)-forms of CaM and TR(2)C with the antipsychotic drug trifluoperazine demonstrated that drug binding to the exposed hydrophobic surfaces in CaM causes substantial conformational changes and proceeds in a sequential manner - first the C-lobe and subsequently the N-lobe. Finally, the field dependence of CaM-bound Pb-207 signals was examined. The Pb-207 Signal linewidths exhibited a sharp dependence on the square of the external magnetic field, a trend characteristic of relaxation via chemical shift anisotropy. Relaxation studies on TR(2)C demonstrated that chemical exchange also contributes to the observed linewidths. The large chemical shift dispersion observed for the Pb-207 signals of the three proteins studied here illustrates the remarkable sensitivity of this parameter to subtle differences in the chemical environment of the protein-bound Pb-207 nucleus. To our knowledge, the data presented in this article comprise the first ever published example of the application of Pb-207 NMR spectroscopy to metalloproteins.