The structures of T₆, T₃R₃ and R₆ bovine insulin: combining X-ray diffraction and absorption spectroscopy

The crystal structures of three conformations, T₆, T₃R₃ and R₆, of bovine insulin were solved at 1.40, 1.30 and 1.80 Å resolution, respectively. All conformations crystallized in space group R3. In contrast to the T₆ and T₃R₃ structures, different conformations of the N-terminal B-chain residue PheB1 were observed in the R₆ insulin structure, resulting in an eightfold doubling of the unit-cell volume upon cooling. The zinc coordination in each conformation was studied by X-­ray absorption spectroscopy (XAS), including both EXAFS and XANES. Zinc adopts a tetrahedral coordination in all R₃ sites and an octahedral coordination in T₃ sites. The coordination distances were refined from XAS with a standard deviation of <0.01 Å. In contrast to the distances determined from the medium-resolution crystal structures, the XAS results were in good agreement with similar coordination geometries found in small molecules, as well as in other high-resolution insulin structures. As the radiation dose for XRD experiments is two orders of magnitude higher compared with that of XAS experiments, the single crystals were exposed to a higher degree of radiation damage that affected the zinc coordination in the T₃ sites in particular. Furthermore, XANES spectra for the zinc sites in T₆ and R₆ insulin were successfully calculated using finite difference methods and the bond distances and angles were optimized from a quantitative XANES analysis.