(24e) Thermodynamics, Structure, and Oligomerization States of Key Intermediates in Non-Native Chymotrypsinogen Aggregate Initiation and Growth

Non-native protein aggregation is a common problem during the production and storage of protein-based pharmaceuticals. It has also been implicated in a number of diseases including Alzheimer's and Parkinson's disease. Although non-native protein aggregation is a significant industrial and biological problem, and much has been learned about it over the past three to four decades, there remain significant outstanding questions about key steps, or equivalently key intermediates, in non-native aggregation pathways. Identification of such intermediates and characterization of their structure and stability is a first step towards strategies to better control aggregation rates and aggregate structures and morphology.

In previous work presented at this meeting, we have shown that α-Chymotrypsinogen A (aCGN) is a convenient model system of non-native aggregation leading to non-fibrillar aggregates. Here we report in detail the process of ?nucleation? and subsequent aggregate growth, utilizing a combination of time-dependent thermal scanning calorimetry and spectroscopy (CD and FL), isothermal and temperature-jumped aggregation kinetics monitored by size-exclusion chromatography, and stopped-flow fluorescence. These results have been used to propose a generalized Lumry-Eyring model that captures the experimentally observed aggregation behaviors.

The results indicate aCGN aggregates through a nucleation-and-growth mechanism under non-native favoring conditions, similar to what has been shown or hypothesized for many fibril-forming proteins. Unlike previous studies, using our approach we are also able to elucidate the following details about key intermediates in the nucleation and growth stages: (1) the initiation or nucleation step proceeds via an enthalpically unfavorable, relatively small, reversible x-mer of denatured monomers; (2) the value of x depends on temperature and protein concentration; (3) this nucleus grows by addition of monomers and/or dimers; (4) the addition of monomers and/or dimers during aggregate growth involves an enthalpically favorable, reversible association that includes a significant gain in β-sheet structure for the added monomer/dimer chain(s); (5) reversible association and rearrangement is followed by a more subtle, irreversible structural conversion that commits the reversibly associated monomers to the irreversible aggregate. To the best of our knowledge, this is the first direct experimental evidence that has shown the underlying secondary structure in the irreversible aggregates is formed upon reversibly binding monomer or dimer to preformed aggregates, rather than the commonly assumed mechanism in which such secondary structure forms as either an equilibrium or transient structure in the native or unfolded monomeric states. By incorporating these newly discovered features into a generalized Lumry-Eyring model of non-native aggregation, we are able identify a set of experimental signatures that may be of utility in identifying key intermediates for other aggregation-prone proteins.