Effects of Protein Unfolding on Aggregation and Gelation in Lysozyme Solutions.

Shakiba Nikfarjam,Taylor J Woehl,Mikhail A Anisimov,Elena V Jouravleva

doi:10.3390/biom10091262

Shakiba Nikfarjam, Taylor J Woehl + Show 2 more

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https://doi.org/10.3390/biom10091262

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Abstract

In this work, we investigate the role of folding/unfolding equilibrium in protein aggregation and formation of a gel network. Near the neutral pH and at a low buffer ionic strength, the formation of the gel network around unfolding conditions prevents investigations of protein aggregation. In this study, by deploying the fact that in lysozyme solutions the time of folding/unfolding is much shorter than the characteristic time of gelation, we have prevented gelation by rapidly heating the solution up to the unfolding temperature (~80 °C) for a short time (~30 min.) followed by fast cooling to the room temperature. Dynamic light scattering measurements show that if the gelation is prevented, nanosized irreversible aggregates (about 10–15 nm radius) form over a time scale of 10 days. These small aggregates persist and aggregate further into larger aggregates over several weeks. If gelation is not prevented, the nanosized aggregates become the building blocks for the gel network and define its mesh length scale. These results support our previously published conclusion on the nature of mesoscopic aggregates commonly observed in solutions of lysozyme, namely that aggregates do not form from lysozyme monomers in their native folded state. Only with the emergence of a small fraction of unfolded proteins molecules will the aggregates start to appear and grow.

Highlights

Aggregation of proteins and peptides is a broad and complex phenomenon with several subsets, each progressing via a distinct mechanism [1]
The role of equilibrium unfolding as the main driving force for protein aggregation at elevated temperatures is clarified
While monomeric lysozyme molecules do not have a propensity to form detectable aggregates for a long period of time [24], increasing the temperature and shifting the equilibrium to marginally unfolded protein leads to the emergence of aggregate seeds

Summary

Introduction

Aggregation of proteins and peptides is a broad and complex phenomenon with several subsets, each progressing via a distinct mechanism [1]. Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, caused by misfolding and aggregation of proteins followed by precipitation of these aggregates in the central nerves system, are examples of indigenous protein aggregation [2]. Because some of the amyloidogenic proteins have a three-dimensional folded structure [4], studying the difference between the driving forces for aggregation of finely folded and partially unfolded proteins would help to elucidate the aggregation mechanisms of pathogenic proteins responsible for amyloid diseases. The increase in the number of protein drugs and the obstacles, caused by aggregation, in production and shelf-life of these drugs is another motivation for studying the aggregation mechanisms [3]

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