X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue.

A crystal structure of a totally inactive insulin molecule has been determined. For this insulin molecule, the first without detectable activity to be characterized, the A and B-chains are linked by a peptide bond between A1 Gly and B29 Lys. The molecule has retained all its normal self-association properties and it can also accommodate the two different conformations designated T and R, as seen in 4Zn native pig insulin crystals. The hexamers of the crosslinked insulin molecule were crystallized using the 4Zn insulin recipe of Schlichtkrull. The structure has been crystallographically refined with data extending to 2 A using restrained least-square methods. Comparison of the B29-A1 peptide crosslink insulin and the 4Zn native insulin reveals close structural similarities with the native dimer. The analysis of the structure confirms the earlier hypothesis that insulin structures in crystals are not in an active conformation and that a separation of N-terminal A-chain and C-terminal B-chain is required for interaction with the insulin receptor.

[1]  E. Dodson,et al.  Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer , 1989, Nature.

[2]  H S Tager,et al.  Role of the phenylalanine B25 side chain in directing insulin interaction with its receptor. Steric and conformational effects. , 1986, The Journal of biological chemistry.

[3]  R. M. Justice,et al.  Complete sequence-specific 1H NMR assignments for human insulin. , 1990, Biochemistry.

[4]  R. Huber,et al.  The Geometry of the Reactive Site and of the Peptide Groups in Trypsin, Trypsinogen and its Complexes with Inhibitors , 1983 .

[5]  Wayne A. Hendrickson,et al.  A restrained-parameter thermal-factor refinement procedure , 1980 .

[6]  E. Dodson,et al.  Structure and biological activity of hagfish insulin. , 1979, Journal of molecular biology.

[7]  J. Helliwell,et al.  Calibration Tests and Use of a Nicolet/Xentronics Imaging Proportional Chamber Mounted on a Conventional Source for Protein Crystallography , 1989 .

[8]  B. Frank,et al.  Interaction of zing with proinsulin , 1970 .

[9]  E. Dodson,et al.  Structural stability in the 4-zinc human insulin hexamer. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[10]  G J Williams,et al.  The Protein Data Bank: a computer-based archival file for macromolecular structures. , 1977, Journal of molecular biology.

[11]  R. Hubbard,et al.  Insulin's structural behavior and its relation to activity , 1983, Biopolymers.

[12]  M. Dunn,et al.  1H Fourier transform NMR studies of insulin: coordination of Ca2+ to the Glu(B13) site drives hexamer assembly and induces a conformation change. , 1988, Biochemistry.

[13]  Tom L. Blundell,et al.  Insulin: The Structure in the Crystal and its Reflection in Chemistry and Biology by , 1972 .

[14]  R. Mirmira,et al.  Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor. Importance of main chain conformation. , 1989, The Journal of biological chemistry.

[15]  G. Dodson,et al.  Rhombohedral insulin crystal transformation. , 1978, Journal of molecular biology.

[16]  D. Hodgkin,et al.  Evidence concerning insulin activity from the structure of a cross-linked derivative. , 1981, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[17]  T L Blundell,et al.  The structure of 2Zn pig insulin crystals at 1.5 A resolution. , 1988, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[18]  L. Thim,et al.  Single chain des-(B30) insulin. Intramolecular crosslinking of insulin by trypsin catalyzed transpeptidation. , 2009, International journal of peptide and protein research.