We are working to develop an appropriate model system for the investigation of polyQ disease-associated protein, and in developing a quantitative description of the kinetics of association of polyQ proteins. Such a framework will facilitate both the identification of the mechanism by which polyQ protein aggregates cause cellular dysfunction, and the development of novel therapeutic strategies to combat the disease.
In this talk we will describe the work we are undertaking with both synthetic polyQ peptides and with recombinant proteins with polyglutamine inserts. We have synthesized polyQ peptides of variable glutamine length and with various flanking residues or imposed structural features. The aggregation properties of these peptides have been tested using a variety of biophysical tools and at several solvent conditions. The data challenge the accepted paradigm that polyQ peptides aggregate via a simple nucleation-elongation mechanism involving a thermodynamically unstable monomeric nucleus. We propose an alternative interpretation of our data along with other published data.
We will also describe ongoing work to develop and characterize a small library of protein mutants containing polyQ inserts. We have chosen apomyoglobin as a model scaffold, and have developed a method for generating a diversity of mutants with polyQ inserts of variable length and insertion location. Currently we are producing these proteins and beginning to characterize their folded structure, stability, and aggregation kinetics. These studies will address questions regarding the role of polyQ in promoting protein aggregation; specifically, is aggregation driven because the polyQ insert disrupts protein folding, or because of specific glutamine-glutamine interactions between proteins?