Nanoskopie

Amyloid fibrils are self-assembled protein aggregates that are formed from unstructured peptides and unfolded proteins. The fibrils are characterized by a universal β-sheet core stabilized with hydrogen bonds, but the molecular structure of the peptide subunits exposed on the fibril surface is variable. Here we show that multimodal spectroscopy using a range of bulk- and surface-sensitive techniques provides a powerful way to dissect variations in the molecular structure of polymorphic amyloid fibrils. As a model system, we use fibrils formed by the milk protein β-lactoglobulin, whose morphology can be tuned by the protein concentration during formation. We investigate the differences in the molecular structure and composition between long, straight and short, worm-like fibrils. We show, using mass spectrometry, that the peptide composition of the two fibril types is similar. The overall molecular structure of the fibrils probed with various bulk-sensitive spectroscopic techniques shows a large contribution of the β-sheet core, but no difference in structure between straight and worm-like fibrils. When probing specifically the surface of the two amyloid types with high spatial resolution using tip-enhanced Raman spectroscopy (TERS), the surface of the fibrils is found to be heterogeneous in molecular structure and mainly exhibits unordered or α-helical structures. The TERS study reveals that the surface of long, straight fibrils contains more β-sheet structure than the surface of short, worm-like fibrils, which is consistent with previous surface-specific vibrational sum-frequency generation (VSFG) spectroscopic results1 that revealed substantially more β-sheet content for the straight than for the worm-like fibrils. In conclusion, the difference in structure between straight and worm-like amyloid fibrils can only be determined using advanced vibrational spectroscopic techniques like TERS and VSFG.