A calbindin D(9k) mutant containing a novel structural extension: 1H nuclear magnetic resonance studies

Abstract

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 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 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.