Amyloid formation is associated with a range of debilitating human disorders including Alzheimer's and prion diseases. The amyloid structure is essential for understanding the role of amyloids in these diseases. Amyloid formation of Ure2 protein underlies the yeast prion [URE3]. Here we use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the structure of amyloid fibrils formed by the Ure2 prion domain. The Ure2 prion domain under study contains a Sup35M domain at C-terminus as a solubilization element. We introduced spin labels at every residue from positions 2-15, and every 5th residue from positions 20-80 in Ure2 prion domain. EPR spectra at most labeling sites show strong spin exchange interactions, suggesting a parallel in-register ? structure. With quantitative analysis of spin exchange interactions, we show that residues 8-12 form the first ? strand, followed by a turn at residues 13-14, and then the second ? strand from residue 15 to at least residue 20. Comparison of the spin exchange frequency for the fibrils formed under quiescent and agitated conditions also revealed differences in the fibril structures. Currently there is a lack of techniques for in-depth structural studies of amyloid fibrils. Detailed structural information is obtained almost exclusively from solid-state NMR. The identification of ?-strand and turn regions in this work suggests that quantitative analysis of spin exchange interactions in spin-labeled amyloid fibrils is a powerful approach for identifying the ?-strand and turn/loop residues and for studying structural differences of different fibril polymorphs.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|