Joseph Bernacki, PhD 2011

Thesis Title: Development of polyalainine peptides and model discrimination analyses as tools for studying protein aggregation

Protein aggregation is a vitally important topic in multiple fields. Mechanistic research into protein aggregation involves kinetic modeling, and model protein aggregation systems are often utilized because many disease-linked proteins are not amenable to in vitro experimentation.

This work presents the development and utilization of two tools which advance the field of mechanistic protein aggregation research. The first involves the development and systematic statistical analysis of a library of mechanistic aggregation models. We demonstrated, via rigorous statistical model discrimination, that many of these models provide equivalently good fits of standard monomer-loss kinetic data. We proved that monomer-loss kinetic data is fundamentally inadequate to discriminate between competing mechanistic models; this is troubling, since monomer-loss kinetic data has become the standard data in the field. We then established the data type and quality necessary for robust determination of the underlying mechanism: multivariate (i.e., monomer loss and aggregate growth) data over the entire aggregation process.

The second tool involves the development of polyalanine peptides. We synthesized polyalanine peptides (Ac-K2 -W-AN -X-K2 -NH2 ) containing uninterrupted polyalanine segments ranging from A6 to A24 . We took care to establish that the peptides began as disaggregated, well-dissolved monomers. We performed multiscale experiments on the peptides and their aggregates, examining both the individual peptide chains (Å-scale) and the extremely large aggregates (μm-scale). All our polyA peptides formed soluble aggregates immediately, without a lag phase. Detailed light scattering analysis of the polyA peptide aggregates revealed that the shorter peptides (A 18 or less) formed loose bundles of aggregates ∼100 nm in size, which coalesced into very large ∼1000 nm structures over the course of hours to weeks. The A 24 peptide aggregates were fundamentally different, forming dense oligomers ∼15 nm in size. These species have never been directly observed before, but are very reminiscent of both oligomers observed experimentally for proteins with An segments and micelles predicted from ab initio solution thermodynamics for A24 peptides.

The methods and results presented in this dissertation have a far-reaching impact on protein aggregation research. The methods and results developed in this work set the stage for exciting developments in pursuit of the mechanism of protein aggregation.