Phase Diagram to Illustrate Protein Aggregation Profile and Conditions

Abstract

Protein self-assembly and formation of amyloid fibers and/or amorphous aggregates is an early event in numerous human diseases, such as Alzheimer's disease, Parkinson's disease, and cataracts. Identification of the structural features generated in the aggregation process, especially under conditions similar to the tissue's viscous and crowded environment, helps to elucidate the mechanism of protein aggregation and the pathogenesis of these diseases. By systematically testing a broad range of conditions and construction of a 3-dimensional phase diagram, we identified the pH, salt, lysozyme concentration, and incubation time, for lysozyme to form amyloid fibers, amorphous aggregates, and gels. We examined the effect of viscosity and molecular crowding on lysozymes' aggregation profile. We characterized the aggregates by use of AFM, TEM, FPLC, and Thioflavin T binding assays, and found that amyloid fibers are formed between pH 2.0 and 3.0, amorphous aggregates at pH 3.5 and above. Glycerol or polyethylene glycol inhibits fiber formation. Gels are formed when fiber concentration is high, and the presence of glycerol or polyethylene glycol lowers the minimum fiber concentration required for gelation. Salt or shaking promotes amyloid fiber formation and shortens the time needed for gelation. Colloidal spheres are present in amyloid fiber solutions, as predicted by the linear colloidal aggregation model we introduced previously. The phase diagrams provide a comprehensive and clear picture of the relation between various aggregates and the conditions for their formation.