Using lanthanide ions to align troponin complexes in solution: Order of lanthanide occupancy in cardiac troponin C

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional {H-1, N-15} HSQC NMR spectroscopy has been used to examine the binding order of Ce3+, Tb3+, and Yb3+ to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce3+, to holochicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca2+, ions in the C-domain binding sites. By using the species cTnC.Yb3+.2Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound N-15-labeled cTnI(129-148) by using two-dimensional {H-1, N-15} inphase antiphase (IPAP) NMR spectroscopy.