Inferring Aggregation Mechanisms of Polyglutamine Through Quantitative Studies of Phase Behavior

Many proteins associated with neurodegenerative diseases are intrinsically disordered, i.e. they lack a stable, folded structure under physiological conditions. It is currently believed that the aggregation of these proteins plays a causative role in neurodegenerative disease pathogenesis. Therefore, understanding how and why these proteins aggregate is crucial to understanding and possibly suppressing disease.Proteins are polymers and protein aggregation is akin to phase separation. We employ techniques and theories borrowed from polymer physics to help understand the driving forces and mechanisms of protein aggregation. Our system of interest is polyglutamine, as expansions of polyglutamine are thought to be causally linked to the development of at least nine different neurodegenerative diseases. In this work, we measure the saturation concentrations of aqueous polyglutamine solutions containing 30 and 40 glutamines and either 2 or 4 lysines. We use classical polymer physics theory to construct the entire (soluble-insoluble) phase diagram from the measured saturation concentrations. The low-concentration arm of the phase diagram provides a thermodynamic basis for assessing aggregation propensity. For a given chain length of polyglutamine, we find that aggregation propensity increases with fewer lysines, and, for a fixed number of lysines, the aggregation propensity increases with increasing chain length. Although the phase diagrams are thermodynamic in nature, they still provide insights regarding the kinetic mechanisms of phase separation. For the concentrations used in most in vitro experiments, the phase diagrams predict that intrinsically disordered monomers first form disordered, higher-order oligomers or clusters which then undergo a nucleated structural conversion into an ordered, insoluble form. These predictions are supported by detailed atomic force microscopy studies. This work highlights the prominence of intrinsic disorder even in multimolecular complexes and its role in facilitating conformational conversion to ordered supramolecular aggregates.