| Insulin Aggregation/amyloid formation of insulin affects the amount of therapeutic dose delivered in insulin pumps designed for efficient insulin delivery in insulin dependent patients. Involvement of the intermediate conformations as precursors or aggregation triggering species has been studied in numerous recent articles. We extended this study to insulin in following two ways: i). Gdn.HCl and urea are the traditional denaturants believed to generate non-native conformations. We first characterized the conformational changes of insulin with these perturbants using CD, SEC and SAXS and then followed the contribution of these structural modofications towards fibrillation under the similar conditions by Thioflavin T fluorescence and Electron microscopy (EM). The studies were carried out at physiological pH (where insulin exists as hexameric) and in 20% acetic acid (where insulin is monomeric). Gdn.HCl at low concentrations dissociated the hexamer novel partially unfolded dimer state, which dissociated into a partially unfolded monomer state at moderate concentrations of Gdn.HCl. The addition of even very low concentrations of Gdn‚HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased (60%) at high concentrations. Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, which existed up to >6 M Gdn‚HCl, accounting for the formation of substantial amounts of fibrils under such conditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was also accelerated by Gdn‚HCl. The enhanced fibrillation of the monomer was due to the increased ionic strength at low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformation at Gdn‚HCl concentrations above 1 M. Moderate concentrations of urea lead to accumulation of a partially unfolded dimer state, which dissociates into an expanded, partially folded monomeric state. Very high concentrations of urea resulted in an unfolded monomer with some residual structure. In the presence of 20% acetic acid, where insulin exists as the monomer, the addition of low concentrations of urea slowed down the rate of fibrillation, e. g. 5-fold at 0.75 M urea. The decreased fibrillation of the monomer was due to an induced non-native conformation with significantly increased a-helical content compared to the native conformation. In 20% acetic acid moderate concentrations of urea accelerated fibrillation kinetics, but high concentrations (>5 M) reduced fibrillation Addition of increasing NaCl concentrations (upto 4M) at constant 2M or 4M urea results in the enhancement of fibrillation with non significant changes in the tertiary structure of insulin at both the hexameric or monomeric form. However even the very small concentration (0.25M) resulted in the dissociation of the oligomeric specie present at 2M 0r 4M urea into monomeric form with considerable changes in the Near UV CD. The fibrillation was again faster even at these very low concentrations of Gdn.HCl. Ahmad, A: Millet, IS: Doniach, S: Uversky, VN: and Fink, AL. (2003) Partially Folded Intermediates in Insulin Fibrillation Biochemistry 42, (in press) Ahmad, A: Millet, IS: Doniach, S: Uversky, VN: and Fink, AL. (2003) Effect of Structural perturbations on insulin fibrillation: Effect Of Urea (submitted to JBC) Ahmad, A: Uversky, VN: and Fink AL. Comparison of contribution of Urea and GdnCl towards insulin structure modification leading to fibrillation. (Manuscript under preparation) ii) Fibrillation of insulin (4mg/mL) was also followed in real time. Aliquots were drawn from the solution, incubated at 37 degree C with mechanical stirring, at various time intervals and analyzed by various techniques viz, CD, ThT and ANS fluorescence, ATR-FTIR, and H/D mass exchange spectroscopy and Electron microscopy. ANS showed changes in protein conformation at 2.5 hrs of incubation before ThT showed the fibril formation. The detection of 10 more hydrogens exchanging at 2.5 hrs of incubation than the native protein in H/D exchange Mass spectroscopy supports the conformational change at 2.5 hrs. The ATR-FTIR studies were used to map the regions of insulin molecule undergoing changes before fibril formation. Based on these observations we propose following pathway. Monomer --> Monomer* --> Fibrils. Ahmad, A: and Fink AL. Characterization of an intermediate conformation on pathway to insulin fibrillation. . (Manuscript under preparation) |