A calbindin D _{1 H nuclear magnetic resonance studies}

Calbindin D_9k is a small, well‐studied calcium‐binding protein consisting of two helix‐loop‐helix motifs called EF‐hands. The P43MG² mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF‐hands (F36‐S44). A lower calcium affinity for P43MG was expected on the basis of simple entropie arguments. However, this is not the case and P43MG (‐97 kJ‐mol⁻¹) has a stronger calcium affinity than P43M (‐93 Kj‐mol⁻¹), P43G (‐95 kJ‐mol⁻¹) and even wild‐type protein (‐96 kj‐mol⁻¹). An NMR study was initiated to probe the structural basis for these calcium‐binding results. The ¹H NMR assignments and ³J_HNHα values of the calcium‐free and calcium‐bound forms of P43MG calbindin D_9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38‐K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium‐bound wild‐type calbindin D_9k‐—but this is the first observation in solution for wild‐type calbindin D_9k or any of its mutants.