NR APIK
AU Jiang,Y.; Li,H.; Zhu,L.; Zhou,J.M.; Perrett,S.
TI Amyloid nucleation and hierarchical assembly of Ure2p fibrils. Role of asparagine/glutamine repeat and nonrepeat regions of the prion domains.
QU The Journal of Biological Chemistry 2004 Jan 30; 279(5): 3361-9
PT journal article
AB The yeast prion protein Ure2 forms amyloid-like filaments in vivo and in vitro. This ability depends on the N-terminal prion domain, which contains Asn/Gln repeats, a motif thought to cause human disease by forming stable protein aggregates. The Asn/Gln region of the Ure2p prion domain extends to residue 89, but residues 15-42 represent an island of "normal" random sequence, which is highly conserved in related species and is relatively hydrophobic. We compare the time course of structural changes monitored by thioflavin T (ThT) binding fluorescence and atomic force microscopy for Ure2 and a series of prion domain mutants under a range of conditions. Atomic force microscopy height images at successive time points during a single growth experiment showed the sequential appearance of at least four fibril types that could be readily differentiated by height (5, 8, 12, or 9 nm), morphology (twisted or smooth), and/or time of appearance (early or late in the plateau phase of ThT binding). The Ure2 dimer (h = 2.6 +/- 0.5 nm) and granular particles corresponding to higher order oligomers (h = 4-12 nm) could also be detected. The mutants 15Ure2 and Delta 15-42Ure2 showed the same time-dependent variation in fibril types but with an increased lag time detected by ThT binding compared with wild-type Ure2. In addition, Delta 15-42Ure2 showed reduced binding to ThT. The results imply a role of the conserved region in both amyloid nucleation and formation of the binding surface recognized by ThT. Further, Ure2 amyloid formation is a multistep process via a series of fibrillar intermediates.
MH Amino Acid Motifs; Amino Acid Sequence; Amyloid/*chemistry/metabolism; Asparagine/chemistry; Dimerization; Glutamine/chemistry; Hydrogen-Ion Concentration; Kinetics; Microscopy, Atomic Force; Molecular Sequence Data; Mutation; Prions/*chemistry/metabolism; Protein Binding; Protein Structure, Tertiary; Saccharomyces cerevisiae Proteins/*chemistry/metabolism; Sequence Homology, Amino Acid; Support, Non-U.S. Gov't; Thiazoles/chemistry; Time Factors
AD National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
SP englisch
PO USA