| abstract |
The invention is a method for in vitro monitoring of peptide or protein fibril assembly. In order to provide sensitivity at nanomolar concentrations on the order of that observed in vivo, the method makes use of fluorescent energy transfer between closely juxtaposed donor and acceptor fluorophores. Accordingly, the invention requires attaching a donor fluorophore to a fibrillogenic peptide or protein, and attaching an acceptor fluorophore to a second fibrillogenic peptide or protein. The donor and acceptor fluorophores are located on the first and second peptides or proteins so that they are juxtaposed to permit energy transfer between them upon fibril formation. The fluorophore-containing peptides or proteins are mixed in solution at a normal physiological concentration, and fibril formation is monitored by observing the fluorescence energy transfer between the donor and acceptor fluorophores. Mixing is preferably done by providing equimolar amounts of peptides or proteins in a denaturant solution, and monitoring of fibril formation is initiated by diluting out the denaturant. |