Year: 2011
Type: article
Abstract: Hexameric insulin exists as an allosteric complex with three well known conformations (T 6 , R 6 and T 3 R 3 ) [1][2][3].Each hexameric complex contains two divalent metal ions (typically Zn).Both ions are located on the three-fold symmetry axis going through the hexamer and coordinate to three symmetry-related histidine N ε2 atoms.Octahedral coordination is fulfilled in the T 6 conformation by further coordination of three water molecules and in the R 6 conformation tetrahedral coordination is fulfilled by coordination of one lyotropic anion, which is also located on the three-fold symmetry axis.A dual octahedral/ tetrahedral coordination is observed in the T 3 R 3 conformation.In this work we have studied all three conformations of bovine insulin by combining complementary techniques: Single crystal Xray diffraction (XRD), X-ray powder diffraction (XRPD) and X-ray absorption fine structure spectroscopy (XAFS).Crystals of T 6 , R 6 and T 3 R 3 zinc insulin were grown and the structures were solved by single crystal XRD, to obtain good model structures for the XAFS data analysis.For bovine insulin only the structure of T 6 conformation has hitherto been solved [4].All three conformations form crystals in space group R3, and can, however, easily be distinguished by XRPD since the unit cell parameters alter.[5] Using in-house XRPD the conformations were verified before and after XAFS experiments.[6] The coordination around the zinc sites were studied by XAFS for all three conformations.Furthermore hexameric T 6 insulin crystallized with copper and nickel were studied.Data were collected on beamline 811 at MAX-lab, Lund, Sweden, and are the first protein XAFS experiments carried out on this beamline.Coordination geometry was verified from the near edge region of the spectra (XANES) by comparison with an octahedral and a tetrahedral zinc imidazole complex, see figure.The coordination geometry was in agreement with the extended region of the spectra (EXAFS) and bond distances to the first coordination shell were determined with uncertainties below 0.02 Å.
Authors: Z. Dauter, M. Dauter, K. Brzezinski, M. Kubicki, M. Jaskolski
Language: English
Subfield: Materials Chemistry
Field: Materials Science
Domain: Physical Sciences
Open Access status: bronze
OpenAlex ID: W2316932507