ABSTRACT
1H-NMR and circular dichroism studies have been carried out on osteocalcin, a 49-residue, calcium-binding protein, the sequence of which contains a disulphide bridge, a proline-rich segment and three gamma-carboxyglutamic acid (Gla) residues. These latter residues have been proposed to lie on one face of an alpha helix and interact with the mineral phase, leading to incorporation of the protein into the bone matrix. Circular dichroism shows an increase in the alpha-helical structure on Ca2+ binding to bovine osteocalcin. This induced structure is lost on heating the protein, giving a spectrum close to that of the Ca(2+)-free protein. 1H-NMR studies of rabbit osteocalcin gave a set of resonance assignments and NOEs which could be interpreted in terms of distance constraints. These did not allow a single conformation to be defined for the protein in solution but reflect rather a flexible structure which may be essential for the function of the protein. The calculated structures contain a hydrophobic core (comprising Leu2, Leu32, Val36 and Tyr42, seen to be slowly flipping in the Ca(2+)-bound form) and have the gamma-carboxyglutamic acid side chains exposed on one face of the molecule.
