NMR investigation and secondary structure of domains I and II of rat brain calbindin 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 located within domain I while domains III, IV, and V bind calcium less tightly. The recombinant protein construct calb I‐II (residues 1–93) comprising the first two 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 4D high‐resolution NMR experiments were applied to calb I‐II, and led to the complete assignment of the 1 H, 13 C and 15 N resonances. The secondary structure of the protein was deduced from the size of the 3 J HN‐Hα coupling constants, the chemical shift indices of 1 Η α , 13 C α , 13 C′ and 13 C β nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major α‐helices are identified: Ala13–Phe23, Gly33–Ala50, Leu54–Asp63, Val76–Leu90, while residues Ala2–Leu6 form a fifth, flexible helical segment. Two short β‐strands (Tyr30–Glu32, Lys72–Gly74) are found preceding helices B and D and are arranged in an anti‐parallel interaction. Based on these data a structural model of calb I‐II was constructed that shows that the construct adopts a tertiary structure related to other well‐described calcium‐binding proteins of the EF‐hand family. Surprisingly, the protein forms a homodimer in solution, as was shown by its NMR characterization, size‐exclusion chromatography and analytical ultra‐centrifugation studies.