Protein Fibrillation Investigated By Fourier Transform Infrared Spectroscopy

Protein fibrillation is a unique type of protein aggregation phenomenon, leading to the formation of protein fibril with a characteristic cross? supramolecular structure. These protein fibrils are also commonly referred to as amyloid fibrils. In vivo, amyloid fibril formation has been associated with dozens of serious human diseases including the well-known Alzheimer's disease and Parkinson's disease. Structural characterization of amyloid fibril is no doubt fundamentally important for the development of therapeutics for amyloid-related diseases. Yet, it is also a very challenging task. In this study, we explored to use side chain-based infrared(IR) probe to gain detailed structural insights into the amyloid fibril by a 21-residue model amyloidogenic peptide, A?(8-28). We first proposed an approach to incorporate thiocyanate(SCN) IR probe in a site-specific manner into amyloidogenic peptide using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate as cyanylating agent. Using this approach, we obtained three A?(8-28) variants, labeled with SCN probe at three different positions, namely, site #9, site #15, and site #26. We then showed with thioflavin T fluorescence assay, Congo red assay, and atomic force microscopy that the three labeled A?(8-28) peptides can quickly form amyloid fibrils under high concentration and under high salt conditions. Finally, we performed a detailed IR spectral analysis of A?(8-28) fibril in both amide I region(1700-1600 cm-1) and SCN probe region(2200-2100 cm-1) and proposed a millipede-like structure for A?(8-28) fibril. In this proposed structure model, A?(8-28) fibril contains two interdigitated ?-sheets. In each ?-sheet, the A?(8-28) peptide chains are arranged in an anti-parallel configuration. Along each peptide chain, the amino acid residues from site #10 to site #26 are in ?-sheet configurations, whereas, the amino acid residues at site #8, site #9, site #27, and site #28 are in disordered states. Intuitively, the whole A?(8-28) fibril looks like a millipede.