PB-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 it s C-terminal tryptic half-molecule (TR(2)C), were analyzed by Pb-207 N MR spectroscopy. For the parvalbumins, two Pb-207 signals were observe d 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 ch emical shift window, could be discerned for CaM. Experiments on TR(2)C permitted the assignment of each signal as due to Pb-207(2+) Occupyin g 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 evidenc e that Pb2+ binding to all four sites occurs simultaneously, in contra st 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 hydroph obic surfaces in CaM causes substantial conformational changes and pro ceeds in a sequential manner - first the C-lobe and subsequently the N -lobe. Finally, the field dependence of CaM-bound Pb-207 signals was e xamined. The Pb-207 Signal linewidths exhibited a sharp dependence on the square of the external magnetic field, a trend characteristic of r elaxation via chemical shift anisotropy. Relaxation studies on TR(2)C demonstrated that chemical exchange also contributes to the observed l inewidths. 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 e nvironment of the protein-bound Pb-207 nucleus. To our knowledge, the data presented in this article comprise the first ever published examp le of the application of Pb-207 NMR spectroscopy to metalloproteins.