Nonnative aggregation of alpha-chymotrypsinogen A and related systems

Nonnative protein aggregation is a type of protein self-assembly in which the resulting soluble or insoluble aggregates are composed of monomers with secondary structures that are significantly different from the native (monomer) state. The presence of even small amounts of nonnative aggregates in protein-based therapeutics may elicit a potentially dangerous immune response or otherwise negatively impact product quality. Nonnative aggregation has also been implicated in a number of debilitating diseases including Alzheimer's and Huntington's. Nonnative aggregation is a complicated, multistep process potentially involving a variety of transient, sparsely-populated intermediates that can be difficult to isolate and characterize experimentally. Despite the collective efforts of many researchers, the factors controlling the formation and properties of nonnative aggregates remain relatively poorly understood.;This dissertation presents a rational approach to investigating nonnative aggregation that is expected to be broadly applicable to a wide variety of systems of biological or pharmaceutical interest. alpha-chymotrypsinogen A is used as the model system for the majority of the work contained herein because at elevated temperatures it readily forms soluble, nonnative aggregates at experimentally tractable timescales that are amenable to interrogation with solution-based scattering, spectroscopic, calorimetric, and chromatographic techniques.;Nonnative aggregates of alpha-chymotrypsinogen A and bovine granulocyte-colony stimulating factor formed under mildly acidic conditions at low ionic strength are characterized using static and dynamic light scattering, circular dichroism, cryogenic transmission electron microscopy and dye binding. While these proteins have very distinct native secondary structures, they form aggregates that are similar in both structure and morphology. Aggregates from both systems are well-described as linear, semiflexible protein polymer chains with relatively low polydispersity. Increasing the initial monomer concentration in solution is shown to result in aggregates of decreasing size.;Insight into the aggregation mechanism of alpha-chymotrypsinogen A is gained by interpreting experimental data in the context of a Lumry-Eyring nucleated polymerization (LENP) model. Two orthogonal approaches are introduced that yield separate nucleation and growth timescales: (i) combining unseeded monomer loss kinetics with static light scattering of the resulting aggregates or (ii) seeded monomer loss kinetics as a function of number concentration of seed. The size of the aggregate nucleus and the stoichiometry of the growth step are also determined. Intrinsic aggregation timescales are obtained by accounting for contributions from the Gibbs free energy of unfolding and the initial protein concentration. The apparent activation energy for the nucleation step is found to be similar to that for folding, while the intrinsic timescale for growth is independent of temperature.;The dissertation concludes by considering the effects of reducing a single native disulfide in alpha-chymotrypsinogen A. Aggregates derived from the reduced form are structurally and morphologically similar to those from the unreduced form, yet the former are stable under solution conditions that would dissociate the latter. This increased resistance could be due to stronger, less solvent-accessible, nonnative interprotein contacts that were formed due to increased local flexibility afforded by removing the disulfide bond. Combining partially-irreversible differential scanning calorimetry data with monomer loss kinetics in the context of the LENP model shows that reducing the native disulfide promotes aggregation both by decreasing the conformational stability of the monomer and by decreasing the size of the aggregate nucleus.