Study of Misfolding and Aggregation for Short Peptide from the Yeast Prion Sup35 Using AFM Imaging and Force Spectroscopy

Abstract

Protein misfolding is a fundamental pathway of protein self-assembly into nanoaggregates of various morphologies. However, our knowledge of this phenomenon is very limited. Protein aggregation is the cause of many conformational diseases justifying further study of protein misfolding phenomenon. We hypothesized earlier that the protein misfolded conformation is characterized by elevated intermolecular forces ultimately leading to aggregation. In this work we studied a short fragment (-GNNQQNY) of the yeast prion Sup35 which is critical for aggregation of the entire protein and presumably in the protein misfolding. By using force spectroscopy we measured forces for the interactions between a single pair of peptides. We established a correlation between aggregate morphology and strength of inter-peptide interactions as well as their pH dependence. The results of this study provide additional support for the importance of single molecule force spectroscopy for elucidating mechanisms of protein misfolding and aggregation. Using AFM imaging we also show that aggregates formed at different conditions (pH) for this short peptide exhibit distinctly different morphologies that cannot be predicted from the kinetics of aggregation study with ThT fluorescence. We found a dramatic difference of fibrils properties and their structure depending on the aggregation conditions. The difference in the aggregate properties was reflected in their adsorption to the surfaces having different properties: negatively or positively charged surface, PEG modified and hydrophobic surface. Salt concentration has also strong influence on kinetics and aggregate morphology with faster kinetics at higher salt concentration. We also show that the replacement of one amino acid residue in the sequence of this short peptide (Q4P) completely abolishes aggregation. Thus, the primary structure of the peptide is a critical determinant of aggregation propensity.