Hydrogen bonding in the carboxyl-terminal half-fragment 78-148 of calmodulin as studied by two-dimensional nuclear magnetic resonance

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Abstract
The C-terminal half-fragment (residues 78-148) of scallop testis calmodulin was investigated by 500-MHz 2-dimensional proton NMR in order to clarify the structure and the structural change accompanying Ca2+ binding. The sequential resonance assignment to individual amino acid residues was made in part (27 out of 71 residues) by a combination of correlated spectroscopy and nuclear Overhauser effect spectroscopy of a 90% H2O solution. In the Ca2+-bound state, resonances of backbone amide protons of Gly-98, Gly-134, Ile-100, Asn-137 and Val-136 appear at extremely low fields. Amide protons of these residues may be H-bonded. In the Ca2+-free state, the amide resonances of Ile-100 and Gly-134 disappear into the crowded normal shift region. This observation indicates that 2 H bonds of Ile-100 and Gly-134 are destroyed (or weakened) as Ca2+ ions are removed from 2 Ca2+-binding sites. Chemical shifts of amide and .alpha.-protons of residues located in the Ca2+-binding loop of domain III are similar to those of domain IV. The conformations of the 2 loops are very similar. The present results can be interpreted in terms of a structure predicted by Kretsinger.