Protein misfolding frequently leads to the formation of supramolecular assemblies known as amyloid fibrils, the deposition of which is associated with over 20 degenerative diseases including dialysis-related amyloidosis, Alzheimer's disease, and prion disease. The amyloid fibril forms via a nucleation-dependent process in which precursor proteins associate slowly to form a nucleus, followed by an extension where the nucleus grows by the sequential incorporation of precursor molecules using the nucleus fibrils as a template. The template-dependent growth is a unique and essential character underlying the propagation of the original structure of fibrils, and consequently, of pathology of amyloidoses. Identifying the molecular mechanisms of the fibril growth is therefore an important challenge to design effective strategies for the inhibition and regulation of amyloidoses.
In this talk, we will present our recent attempts to monitor a monomer-fibril complex of beta2-microglobulin formed transiently during the fibril growth. By using changes in tryptophan fluorescence spectrum, we analyzed kinetics of the seed-dependent reaction at different concentrations of seeds. While the reaction proceeded in a single-exponential manner at low concentrations of seeds, we observed the accumulation of the fibril-monomer complex in the early stage of the reaction at excessive concentrations of seeds. Mutational analysis suggested that the region around Trp60 is one of the possible interfaces with the fibril. Furthermore, we have developed hydrogen/deuterium (H/D) exchange of the fibril-monomer intermediate. Under the accumulating condition of the fibril-monomer complex, H/D exchange was performed by competitive exchange or by quenched-flow exchange, and proton occupancy of each residue was analyzed by 1H-15N HSQC spectrum, which has provided detailed information about the intermediate structure.
