The objective of this research project is to identify, through molecular modeling techniques, phenolic derivatives which will bind to the insulin hexamer and stabilize an alpha-helical conformation in residues Bl-B8 of the B-chains. The presence of this alpha-- helical conformation reduces zinc exchange and extends the duration of action in certain therapeutic insulin preparations. Monoclinic insulin crystals, grown in the presence of phenol, contain hexamers which adopt the conformation described above. The results of spectroscopic studies of insulin solutions containing phenol have been interpreted on the basis of the monoclinic structure and suggest further that the presence of phenol may produce longer acting forms than are currently available. Crystallographic results from the monoclinic form show that the observed alpha- helical conformation is stabilized by the presence of six phenol molecules within the insulin hexamer. Each phenol molecule resides in an ellipsoidal cavity where if forms two hydrogen bonds to A- chain residues, A6 and All, of an adjacent insulin monomer. The observed spatial requirements and the nature of the hydrophobic and hydrophilic surfaces within these phenol binding sites will be the basis for molecular modeling studies to identify potential binding agents. The ability of these reagents to form complexes and stabilize the alpha-helical conformation will bs verified by crystal growth experiments. Crystallization of the monoclinic form, identified by X-ray diffraction techniques, is indicative that binding has occurred and that the desired conformation has been produced. The threshold concentration of the proposed reagents which is required to effect complexation will also be determined. At the same time, the crystallographic refinement of the monoclinic form of human insulin will be completed (R = 0.22 for 28,119 data to a resolution of 1.8 angstrom) and a crystal structure determination will be initiated on the symmetric R32 form of insulin, also grown in the presence of phenol. Structural studies of insulin have been used in the past to explain known physical properties of insulin preparations, such as the fast- and slow-acting aspects of insulin section. The opportunity nov exists to use new structural information to design reagents which will improve the properties of therapeutic insulin preparations. This will ultimately result in the better management of diabetes and will improve the life style of the diabetic patient.
| Smith, G D; Ciszak, E; Pangborn, W (1996) A novel complex of a phenolic derivative with insulin: structural features related to the T-->R transition. Protein Sci 5:1502-11 |
| Smith, G D; Ciszak, E (1994) The structure of a complex of hexameric insulin and 4'-hydroxyacetanilide. Proc Natl Acad Sci U S A 91:8851-5 |
| Ciszak, E; Smith, G D (1994) Crystallographic evidence for dual coordination around zinc in the T3R3 human insulin hexamer. Biochemistry 33:1512-7 |
| Smith, G D; Dodson, G G (1992) Structure of a rhombohedral R6 insulin/phenol complex. Proteins 14:401-8 |
| Smith, G D; Dodson, G G (1992) The structure of a rhombohedral R6 insulin hexamer that binds phenol. Biopolymers 32:441-5 |
