Structural and biophysical characterisation of repetitive bacterial surface proteins

Protein aggregation is a significant issue both clinically and in the development of protein-based pharmaceuticals and biotechnology. Aggregation occurs when a partially or fully unfolded protein misfolds to form a non-native fold that self-associates into a non-functional oligomeric species. Multi-domain proteins have evolved under a selective pressure to avoid inter-domain misfolding. In proteins with tandemly arrayed domains inter-domain sequence identity is usually less than 50%. Lower sequence identity is thought to avoid the formation of native like inter-domain misfolds. However, in Gram-positive bacteria, a family of proteins have been identified that contain tandemly arrayed repeat domains with between 80-100% sequence identity. It is hypothesised that these domains will be highly stable and aggregation resistant. The aim of this project was to structurally and biophysically characterise domains from a repetitive context and to assess if they exhibit the predicted properties of stability and aggregation resistance. Structures of three high identity tandem repeat domains, Rib Long, Rib Standard and SasYr, are reported. Rib Long and Rib Standard present an example of domains in the same protein where a loss of a core secondary structure has led to a significant increase in thermal stability. The SasY repeat domain was solved by NMR spectroscopy and identified as an Ig-like fold. The thermal stability and aggregation resistance of six high identity repeats was compared to a low identity repeat and a non-tandem domain. High identity repeat domains were found to be highly aggregation resistant and have reasonably high refolding efficiency even after ten cycles of thermal denaturation and refolding at a high concentration. The effects of heat cycling were studied and discussed in context to the structures of the domains. The results presented here will be useful in the design of highly stable and aggregation resistant proteins for use in the pharmaceutical and biotechnology industries