Monitoring the earliest amyloid-beta oligomers via quantized photobleaching of dye-labeled peptides.
Misfolding and aggregation of amyloid-beta peptide (Abeta) are widely recognized as causative events in Alzheimer's disease (AD). Contrary to earlier hypotheses, recent studies have identified soluble Abeta oligomers as the pathogenic agents and documented neurodegenerative effects from species as small as dimers and trimers. As such, detection and characterization of the earliest Abeta oligomers are paramount to understanding, preventing, and treating AD. We have exploited quantized photobleaching from individual dye-labeled Abeta peptides to characterize monomers and small oligomers tethered to the surface of functionalized coverslips. In this way, we have directly determined reproducible distributions of peptide species in various sample environments. Fresh samples (30pM peptide) at pH 7.4 consist primarily of monomers and dimers. Both acidic conditions (pH 5.8) and zinc coordination promote greater association, which is further increased in samples prepared from more concentrated stocks. Acid- or zinc-promoted association is largely prevented and/or reversed by addition of the beta-sheet breaker peptide iAbeta5 or the chelator clioquinol, respectively. These results are qualitatively consistent with bulk-solution studies of Abeta40 and demonstrate a powerful approach for definitively characterizing Abeta oligomers. The methodology utilized here holds promise for assessing aggregation promoters and inhibitors and investigating peptide association in its earliest stages.
Dukes
Kyle D
KD
Department of Chemistry, Physics, and Geology, Winthrop University, Rock Hill, SC 29733, USA.
Rodenberg
Cassandra F
CF
Lammi
Robin K
RK
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D038761:Photobleaching; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D019809:Streptavidin / Q000378:metabolism; D013997:Time Factors
D016229:Amyloid beta-Peptides; D005456:Fluorescent Dyes; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); D001710:Biotin; D019809:Streptavidin