AbstractAs a posttranslational modifier, polyubiquitin is involved in the regulation of diverse intracellular processes; however, it is also found in pathological protein aggregates associated with Alzheimer’s disease and other neurodegenerative disorders. We previously observed that various types of polyubiquitin can form amyloid-like fibrils; however, the structural properties of these polyubiquitin fibrils have not been examined at an atomic level. Here we demonstrate that a soluble intermediate species can be extracted from disulfide-conjugated diubiquitin fibrils after cleaving the disulfide bonds in the fibrils. This newly discovered molecule is structurally and physicochemically distinguishable from native ubiquitin. In addition, it is thermodynamically metastable, as demonstrated by real-time NMR measurements. Collectively, our results suggest that the fibril-derived molecule is a minimal building block of polyubiquitin fibrils that reflects their structural and physicochemical properties.