Understanding the Role of Chain Flexibility in Amyloid Protein Aggregation through Rationally Designed Protein Sequences

Abstract

The aggregation of amyloid proteins is associated with a myriad of medical conditions including Alzheimer's disease (AD), diabetes, and Parkinson's disease. While attributable to different amyloid proteins, these proteins all share a common feature: a periodic glycine motif (GxxxG). This glycine motif, associated with increased backbone flexibility, is extended in a number of familial mutations that significantly increase severity of AD. A better understanding of the role that chain flexibility plays in protein aggregation will allow for new and innovative protein engineering strategies for nanotech development as well as give insight into therapeutic strategies.In this study, the glycine motif is targeted via either extension, by introduction of additional periodic glycine, or contraction, by replacement of glycine with a bulky or constrained amino acid. Modifications that extend periodic glycine include those that align with familial AD mutations. To examine how these alterations to the glycine repeat motif impact aggregation kinetics, aggregation was monitored via thioflavin-T fluorescence and fit using a novel kinetic equation that accounts for unique features observed at late stages of aggregation. Aggregation products were visualized using transmission electron microscopy to examine morphological features. In addition, aggregates were fractionated by size exclusion chromatography for size analysis via light scattering and morphology analysis via surface hydrophobicity. Results indicate that increased chain flexibility correlates with faster nucleation as well as a larger quantity of small, intermediate aggregates that exhibit reduced fibril morphology with unchanged surface hydrophobicity. Taken together with observations that smaller aggregates are more physiologically active, these results support the hypothesis that increases in protein chain flexibility, including that associated with familial mutations, may contribute to disease progression.Future work will extend studies to other amyloid proteins, such as amylin and chaplin H.