Ure2 is the protein determinant of the [URE3] prion state in Saccharomyces cerevisiae,and it forms amyloid fibrils in vitro via conformational change.At the early stages of amyloid fibril assembly,oligomeric intermediates appear as the crucial species for the nucleation process,which are difficult to study due to their heterogeneous and frequently transient nature.However,single-molecule techniques have been developed to observe the structural details of individual intermediate oligomers,such as single-molecule fluorescence resonance energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS).Here,we studied the conformational changes of Ure2 during amyloid assembly using smFRET.We found that N-terminal labeled native Ure2 dimers aggregated to form two different groups of species with different size and FRET distributions--the smaller ones showed high FRET and the larger ones showed an additional lower FRET component,suggesting that the two kinds of species may have different conformations and undergo different assembly pathways.Both the ensemble FRET and smFRET study showed that the FRET of C-terminal domain labeled Ure2 did not change before and after fibril formation while that of the N-terminal labeled Ure2 changed obviously,suggesting that the C-terminal domain of Ure2 remains in the native dimeric form in the fibril.