NMR investigation and secondary structure of domains I and II of rat braincalbindin D-28k (1-93)

Calbindin D-28k, a member of the troponin C superfamily of calcium-binding proteins, contains six putative EF hand domains but binds only four calcium -atoms: one at a binding site of very high affinity and three calcium-atoms at binding sites of lower affinity. The high-affinity site could be locate d within domain I while domains III, IV, and V bind calcium less tightly. T he recombinant protein construct calb I-II (residues 1-93) comprising the f irst tale EF hands affords a unique opportunity to study a pair of EF hands with one site binding calcium tightly and the second site empty. A series of heteronuclear 2D, 3D and 3D high-resolution NMR experiments were applied to calb I-II, and led to the complete assignment of the H-1, C-13 and N-15 resonances. The secondary structure of the protein was deduced from the si ze of the (3)J(HN-H alpha) coupling constants, the chemical shift indices o f H-1(alpha), C-13(alpha), C-13(i) and C-13(beta) nuclei and from an analys is of back;bone NOEs observed in 3D and 3D NOESY spectra. Four major cx-hel ices are identified: Ala13-Phe23, Gly33-Ala50, Leu54-Asp63, Val76-Leu90, wh ile residues Ala2-Leu6 form a fifth, flexible helical segment. Two short be ta-strands (Tyr30-Glu32, Lys72-Gly74) are found preceding helices B and D a nd are arranged in an anti-parallel interaction. Based on these data a stru ctural model of calb I-II was constructed that shows that the construct ado pts a tertiary structure related to other well-described calcium-binding pr oteins of the EF-hand family. Surprisingly, the protein forms a homodimer i n solution, as was shown by its NMR characterization, size-exclusion chroma tography and analytical ultra-centrifugation studies.