An intersheet packing interaction in A beta fibrils mapped by disulfide cross-linking.
Most models for the central cross-beta folding unit in amyloid fibrils of the Alzheimer's plaque protein Abeta align the peptides in register in H-bonded, parallel beta-sheet structure. Some models require the Abeta peptide to undergo a chain reversal when folding into the amyloid core, while other models feature very long extended chains, or zigzag chains, traversing the protofilament. In this paper we introduce the use of disulfide bond cross-linking to probe the fold within the core and the packing interactions between beta-sheets. In one approach, amyloid fibrils grown under reducing conditions from each of three double cysteine mutants (17/34, 17/35, and 17/36) of the Abeta(1-40) sequence were subjected to oxidizing conditions. Of these three mutants, only the Leu17Cys/Leu34Cys peptide could be cross-linked efficiently while resident in fibrils. In another approach, double Cys mutants were cross-linked as monomers before aggregation, and the resulting fibrils were assessed for stability, antibody binding, dye binding, and cross-seeding efficiency. Here too, fibrils from the 17/34 double Cys mutant most closely resemble wild-type Abeta(1-40) fibrils. These data support models of the Abeta fibril in which the Leu17 and Leu34 side chains of the same peptide pack against each other at the beta-sheet interface within the amyloid core. Related cross-linking strategies may reveal longer range spatial relationships. The ability of the cross-linked 17/35 double Cys mutant Abeta to also make amyloid fibrils illustrates a remarkable plasticity of the amyloid structure and suggests a structural mechanism for the generation of conformational variants of amyloid.
Shivaprasad
Shankaramma
S
Graduate School of Medicine, University of Tennessee, 1924 Alcoa Highway, Knoxville, Tennessee 37920, USA.
Wetzel
Ronald
R
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D000911:Antibodies, Monoclonal / Q000378:metabolism; D052160:Benzothiazoles; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D010449:Peptide Mapping / Q000379:methods; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000378:metabolism
D016229:Amyloid beta-Peptides; D000911:Antibodies, Monoclonal; D052160:Benzothiazoles; D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D013844:Thiazoles; C078592:amyloid beta-protein (1-40); C009462:thioflavin T; D003545:Cysteine