Spectroscopic Evidence of Tertiary Structural Differences Between Insulin Molecules in Fibrils

Abstract

Protein fibrils are of great interest due to their involvement in several pathologies and their roles in the degradation of many therapeutic protein products. Fibrils share highly similar secondary structural motifs across different proteins and applied stress conditions. However, fibril morphology differs according to the surrounding conditions, with aromatic and hydrophobic amino acids playing important roles in mature fibril formation. In this study, we use Raman microscopy, by means of the aromatic amino acids in insulin molecules as markers, to probe for tertiary structure differences within fibrils. We compared 2 different fibril types, linear fibril bundles and spherulites. Generation of linear fibril bundles was undertaken in an acetic acid-containing formulation, whereas spherulites were generated in a hydrochloric acid-containing formulation. The Raman intensities of tyrosine and phenylalanine side chains suggest that there are significant differences between the fibril bundles. The findings suggest that the insulin components of the fibril strands are not arranged identically in the 2 fibril types and that this gives rise to differences in their tertiary structures. Overall, the work indicates that the physicochemical properties of fibril structures can be altered by changing the formulation and that these alterations can be monitored by Raman spectroscopy.

Keywords: Raman spectroscopy; insulin; microparticle(s); protein aggregation; protein folding.