Structure and Assembly of Functional Amyloids

The ability of proteins to self‐assemble into beta‐sheet‐rich aggregates called amyloid fibrils is considered to be universal, although certain polypeptide sequences have a particularly high propensity to adopt these conformations. In many cases the formation of amyloid fibrils is deleterious and associated with the progression of disease, but there are also examples of proteins for which the cross‐beta structure represents the functional conformation. Ure2 is the protein determinant of the yeast prion [ URE3 ]. Ure2 consists of an N‐terminal prion‐inducing domain that is disordered in the native state, whereas the C‐terminal functional domain has a globular fold with structural similarity to glutathione transferase enzymes. The C‐terminal domain shows enzymatic activity in both the soluble and fibrillar forms of Ure2. We have used a variety of biophysical approaches to investigate the structure of Ure2 fibrils and their mechanism of assembly. We have also created chimeric constructs where the prion domain is genetically fused to other enzymes of different sizes and architectures. These chimeric polypeptide constructs spontaneously self‐assemble into nanofibrils with fused active enzyme subunits displayed on the amyloid fibril surface. We can measure steady‐state kinetic parameters for the appended enzymes in situ within fibrils, and compare these for the identical protein constructs in solution. We are currently investigating the use of microfluidic techniques to form enzymatically‐active microgel particles from the chimeric self‐assembling protein nanofibrils. These approaches allow amyloid fibrils to be used as a scaffold for enzyme immobilization, facilitating recycling and reuse of the appended enzymes in functional assays, both in batch processes and for microflow chemistry.