Biophysical characterization of folding and aggregation behavior in model single- and multi-domain proteins

Date
2016
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University of Delaware
Abstract
The growing use of protein therapeutics to treat a number of disease states necessitates a thorough understanding of the biophysical properties of these molecules in order to deliver a stable and effective product in a cost-effective manner. Degradation pathways, such as misfolding or non-native aggregation, represent a major threat to product quality and safety and are of considerable interest to the biopharmaceutical industry both for recombinant folding applications and product shelf life concerns. Although folding and product stability are treated as separated challenges, they are both driven by the same biophysical forces and are interrelated. This dissertation investigates the relationship between protein folding and non-native aggregation using three different model protein system to investigate complementary aspects of this relationship to understand how properties of the monomer can be used to characterize and mitigate aggregation. ☐ Reproducible non-native aggregates created from the model protein α-chymotrypsinogen (aCgn) were used to characterize how aggregates can evolve when exposed to environmental conditions that alter the folding equilibrium of the monomeric subunits (i.e. high temperature or denaturant concentration). Aggregates created at elevated temperatures (> 75°C) displayed similar morphologies to control cases, but were more resistant to chemical dissociation. Analogous behavior was observed for control aggregates incubated in non-dissociating concentration of urea prior to removal of the denaturant; these aggregates then required more chemical driving force to induce dissociation. From these results, it was hypothesized that shifting the folding equilibrium to slightly favor the denatured states allows aggregate subunits to reorganize to more “stable” configurations, in a process termed annealing. A theoretical two-dimensional energetic landscape was proposed to describe the annealing process and define the aggregates species in the context of the monomer equilibrium. Despite increased resistance to chemical and thermal denaturation, no distinct spectroscopic signal was detected to differentiate these aggregates and identify the structural changes to the aggregate that impart increased stability. ☐ The conformational stability and folding behavior of was investigated for the engineered single chain antibody fragment, 4-4-20-6His. This system served a model two-domain protein with strong inter-domain interactions for the purpose of understanding how properties of the monomeric protein can be applied to irreversible processes such as aggregation. Although this portion of the work was limited due to material constraints, a baseline biophysical characterization across a range of pH (6.5 – 8.5) and salt conditions (0 – 100 mM) provided insight into the potential complexity of the folding pathway. The cooperative unfolding transition displayed in thermal and chemical unfolding initially appeared to fit a two-state approximation, but this approach was unable to fully describe the observed data and suggested that a weakly-populated intermediate could lead to irreversible degradation pathways. ☐ Finally, ensemble-averaged biophysical characterization techniques were used to characterize a five-domain recombinant immunotoxin, m. pasudotox, and understand the folding behavior of the monomer so as to mitigate off-pathway degradation during refolding. The thermal and chemical unfolding behavior was assessed across a pH window (pH 6.0 – 8.0) and displayed two distinct unfolding transitions (i.e. pseudo-three-state folding model). The identity of the domains involved in these transitions was indirectly confirmed through measurements with the nucleotide NAD+. The two effective transitions, corresponding to the toxin domains and to the antibody domains, displayed significantly different conformational stabilities and folding rates. A stepwise refolding process was designed based on this biophysical characterization to prevent simultaneous folding of the two effective domains and limit inter-domain interactions, and resulted in a marked decrease in soluble aggregates.
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