A CALBINDIN D-9K MUTANT CONTAINING A NOVEL STRUCTURAL EXTENSION - H-1NUCLEAR-MAGNETIC-RESONANCE STUDIES

Calbindin D-9k is a small, well-studied calcium-binding protein consis ting of two helix-loop-helix motifs called EF-hands. The P43MG(2) muta nt 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 simp le entropic 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 the se calcium-binding results. The H-1 NMR assignments and (3)J(HNH alpha ) values of the calcium-free and calcium-bound forms of P43MG calbindi n D-9k mutant are compared with those of P43G. These comparisons revea l 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 stabil ize this part of the protein structure for P43MG(Ca). Several characte ristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calc ium-bound wild-type calbindin D-9k-but this is the first observation i n solution for wild-type calbindin D-9k or any of its mutants.