Total protein synthesis: a new tool for studies of the structure and stability of amyloid fibrils

Amyloid diseases include many well known conditions such as Alzheimer's disease and new variant Cruetzfeld-Jakob disease. All these conditions involve the aggregation of protein molecules into large insoluble fibrils. The purpose of this research is to understand how proteins assemble into amyloid fibrils at the molecular level. We aim to achieve this by altering the structure of beta-2-microglobulin, a protein involved in an amyloid condition that affects all people on long-term renal dialysis, and to investigate how the changes to the protein sequence alter the ability of the protein to form amyloid fibrils. In this particular study we will make changes to the fundamental backbone structure of the protein and such manipulations can not be made using biochemical methods. Instead we will synthesise the whole protein structure using chemistry making each amide bond in a laboratory reactor. The chemical approach does not have the same limitations as biochemistry and so we can make profound changes to the fundamental structure of the target protein. By studying how these changes to the protein structure alter its ability to form amyloid fibres we will elucidate new information about the structure and stability of amyloid fibrils that will inform future work to develop new therapies against these fatal diseases.

Total chemical synthesis offers exciting new opportunities to study protein structure, stability and assembly mechanisms since sequences can be created that are not limited by the specificities of biosynthesis. Despite its potential, however, this approach has not been used widely to date, principally because the synthetic routes are far from routine. Here we propose to use and optimise total chemical synthesis to produce novel sequences of the 99 residue protein, beta-2-microglobulin, an important protein that is the causative agent of a human amyloid disease. By modifying the backbone structure to include two or more N-methylated amino-acids at specific sites, we will quantify the role of the hydrogen bond in determining the stability of the monomeric, native protein as well as the amyloid fibrils formed. The data generated will provide the first quantitative measurements of the role of the main-chain in amyloid structure and assembly. In addition, by combining the results obtained with those from cross-linking studies, using fibrils incorporating non-natural amino acids with photoactivatable side-chains also introduced by total synthesis, we will determine residue-specific restraints that will enable various models for the molecular architecture of beta-2-microglobulin amyloid to be distinguished and,if necessary, new models generated.