Quantitative analysis of the time course of Aβ oligomerization and subsequent growth steps using tetramethylrhodamine-labeled Aβ

Kanchan Garai, Carl Frieden

Research output: Contribution to journalArticle

89 Scopus citations

Abstract

Although amyloid β (Aβ) is a critical player in the pathology of Alzheimer's disease, there is currently little Information on the rate and extent of formation of oligomers that lead to the presence of Aβ fibrils observed in amyloid plaques. Here we describe a unique method to monitor the full time course of Aβ aggregation. In this method, Aβ is labeled with tetramethylrhodamine at a lysine residue on the N-terminal end. During aggregation, the fluorescence is quenched in a time-dependent manner in three distinct phases: an early oligomerization phase, an intermediate phase, and a growth phase. The oligomerization phase can be characterized as a monomer-dimer-trimer process for which we have determined the rate and equilibrium constants. The rate constants differ markedly between Aβ1-42 and Aβ1-40, with Aβ1-42 showing a greater oligomerization propensity. The intermediate phase reflects slow clustering and reorganization of the oligomers, whereas the growth phase ultimately results in the formation of fibrillar material. The data are consistent with a conformational change being an important rate-limiting step in the overall aggregation process. The rates of all phases are highly sensitive to temperature and pH, with the pH-dependent data indicating important roles for lysine and histidine residues. From the temperaturedependent data, activation energies of oligomerization and fibrillization are estimated to be 5.5 and 12.1 kCal/mol, respectively. The methodologies presented here are simple and can be applied to other amyloidogenic peptides or proteins.

Original languageEnglish
Pages (from-to)3321-3326
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number9
DOIs
StatePublished - Feb 26 2013

Keywords

  • Fluorescence quenching
  • Kinetics of aggregation
  • Nucleation
  • Oligomer formation

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