Bovine Serum Albumin protofibril-like aggregates formation: Solo but not simple mechanism

We report an experimental study on the model protein Bovine Serum Albumin (BSA), with the aim of elucidating the mechanisms by which a fully folded globular protein undergoes different aggregation pathways leading to the formation of amyloid fibrils or amorphous aggregates. We observe thermally induced formation of fibrillar structures at pH far from the protein isoelectric point. The increase of electrostatic repulsion results in protein destabilization and in modifications of inter and intra-molecular interactions leading to the growth of fibril-like aggregates stabilized by inter-molecular-β sheets. The aggregation kinetics is studied by means of fluorescence techniques, light scattering, Circular Dichroism (CD), infrared spectroscopy (FTIR) and Atomic Force Microscopy (AFM). Changes in protein secondary structures turn out to be the driving mechanism of the observed aggregation and they progress in parallel with the growth of Thioflavin T emission intensity and scattering signal. This concurrent behavior suggests a mutual stabilization of elongated protofibril-like structures and of protein conformational and structural changes, which lead to a more rigid and ordered structures. Our results give new insights on BSA self-assembly process in alkaline conditions clearly providing new pieces of evidences of the interplay of several and interconnected mechanisms occurring on different time and length scales.

Sketch of BSA aggregation pathways: temperature increase induces partial unfolding of the native structure and the consequent exposure of reactive areas that may produce aggregation processes. At pH values close to isoelectric point, due to the lack of electrostatic repulsion, amorphous aggregates are formed via non-specific interactions mainly of hydrophobic nature. At pH values away from isoelectric point, larger repulsion between molecules slows down the aggregation favoring the structural reorganization of the protein and the formation of b-aggregate structures.Figure optionsDownload high-quality image (96 K)Download as PowerPoint slideResearch highlights
► We study the molecular mechanisms involved in BSA protofibril formation.
► We observe a non-nucleated regular growth of single protofibril-like species.
► Intermolecular β-aggregates formation is the solo supramolecular assembly mechanism.
► Lifetime data show the reduction of conformational heterogeneity of fibrillar state.
► BSA aggregation mechanisms and their interplay are regulated by solution conditions.