(228eg) Effect of Subtle Differences in Amino Acid Sequences on the Aggregation of Insulin Fragments
Insulin aggregates at low pH and high temperature. The aggregates have a signature intermolecular beta sheet structure which is a characteristic of amyloid structures. Insulin is a highly conserved hormone exhibiting minor differences in the amino acid sequence from species to species. For example, human and bovine insulin differences are limited to four amino acids. In a previous study we found that human and bovine insulin exhibit very different aggregation kinetics. Both lag times and rate constants were different. Moreover, dynamic light scattering and FTIR experiments showed that both insulins seem to follow different aggregation pathways as shown by different populations of aggregate sizes and different secondary structure change. Still, mature fibrils were practically identical. To advance our knowledge about this we decided to study fragments of these two insulins that contain the sequence where the differences exist. The fragments were synthesized in solid phase using Fmoc chemistry. Liquid/gas interfaces were provided by stirring whereas solid/liquid interfaces were provided by adding liposomes of different chemistries. Fibrillation was studied as a function of pH, temperature and the presence of air/liquid and liquid/solid interfaces. An experimental design was made to determine the main factors that contribute to fibrillation. A Thioflavin T fluorescence assay was performed to monitor the time evolution of the aggregation process. FT-IR (Fourier Transform Infrared Spectroscopy) was used to monitor the changes in secondary structure and the presence of fibrils was further confirmed by transmission electron microscopy. The exploratory experiments showed differences in the kinetics of both fragments with the bovine one aggregating faster; the same trend is observed with the entire hormone. The best conditions were pH 2.5, a temperature of 60°C under stirring conditions. These conditions were used to further explore the differences between the aggregation phenomena of both fragments by increasing the sampling time and by including DLS measurements. Different mutations in the non-conserved amino acids were explored to evaluate the aggregation mechanism.