Our focus is on examining the conformational changes that may occur in the insulin molecule, in both its hexameric and monomeric states, via the use of synthetic analogs. Research was undertaken by the preparation of analogs with the substitution of phenylalanine-324, phenylalanine-B25, or tyrosine-B26 by tryptophan in the C-terminal region of the B-chain of insulin. These tryptophan residues would later serve as probes for fluorescence spectroscopic analysis. Visible spectroscopic analysis of native insulin was initially undertaken to determine if cyclohexanol could stabilize a characteristic tense to relaxed (T to R) conformational change in the molecule that has been observed using phenol. This change consists of the formation of an alpha-helical region in the N-terminal B-chain domain that occurs concomitant with changes in the geometries of the cobalt coordination centers in the hexameric form of insulin. Cyclohexanol did in fact prove to stabilize this transformation and was used in related experiments in which the T to R transformation of tryptophan-containing analogs were observed by fluorescence spectroscopy. interestingly the conformational changes that were observed also appear to occur in the monomeric forms of the analogs. Further studies will be conducted to localize and characterize the structural changes occurring during the T to R transition of insulin, and evaluate the implications of insulin structure in its biologic actions.
| Pittman 4th, I; Nakagawa, S H; Tager, H S et al. (1997) Maintenance of the B-chain beta-turn in [GlyB24] insulin mutants: a steady-state fluorescence anisotropy study. Biochemistry 36:3430-7 |
