N-15 NMR ASSIGNMENTS AND CHEMICAL-SHIFT ANALYSIS OF UNIFORMLY LABELED N-15 CALBINDIN D9K IN THE APO, (CD2+)1 AND (CA2+)2 STATES

N-15 has been uniformly incorporated into the EF-hand Ca2+-binding protein calbindin D9k so that heteronuclear experiments can be used to further characterize the structure and dynamics of the apo, (Cd2+), and (Ca2+)2 states of the protein. The N-15 NMR resonances were assigned by 2D N-15-resolved H-1 experiments, which also allowed the identification of a number of sequential and medium-range H-1-H-1 contacts that are obscured by chemical shift degeneracy in homonuclear experiments. The N-15 chemical shifts are analyzed with respect to correlations with protein secondary structure. In addition. the changes in N-15 chemical shift found for the apo-->(Cd2+)1-->(Ca2+)2 binding sequence confirm that the effects on the protein are mainly associated with chelation of the first ion.