Probing Alzheimer amyloid peptide aggregation using a cell-free fluorescent protein refolding method.
Fibrillation of the Alzheimer beta-amyloid peptide (Abeta) and (or) formation of toxic oligomers are key pathological events in Alzheimer's disease. Several strategies have been introduced to identify small molecule aggregation inhibitors, and based on these methods, a number of aggregation inhibitors have been identified. However, most of these methods use chemically synthesized Abeta42 peptides, which are difficult to maintain in a monomeric state at neutral pH where anti-aggregation screening is usually carried out. We have developed a cell-free Abeta42 aggregation assay based on fluorescence protein refolding. This assay utilizes nanomolar concentrations of protein. We genetically fused Abeta42 to the N-terminus of vYFP, a variant of of GFP, and expressed and purified the folded fusion protein. The refolding efficiency of Abeta42-vYFP fusion was inversely correlated with the solubility of Abeta42. Using fluorescence to monitor refolding of Abeta42-vYFP, we confirmed that Zn2+ binds to Abeta42 and increases its aggregation. The IC50 value estimated for Zn binding is 3.03 +/- 0.65 micromol/L. We also show that this technique is capable of monitoring the aggregation of chemically synthesized Abeta42.
Arslan
Pharhad Eli
PE
Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 101 College Street, Toronto, ON M5G 1L7, Canada.
Chakrabartty
Avijit
A
D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D015335:Molecular Probes; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000737:chemistry
D016229:Amyloid beta-Peptides; D001426:Bacterial Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D015335:Molecular Probes; D010446:Peptide Fragments; C054562:yellow fluorescent protein, Bacteria; D003300:Copper; D015032:Zinc