Multimodal spectroscopic study of amyloid polymorphism 3

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 overall structure of amyloid fibrils is variable. The milk protein β-lactoglobulin forms amyloid fibrils upon incubation at low pH and high temperature. It is possible to tune the morphology and rigidity of the fibrils by the protein concentration during formation. We investigated 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 fibrils types is similar. The bulk molecular structure of the fibrils probed with various spectroscopic techniques shows a large contribution of the β-sheet core, but no difference in structure between straight and worm-like fibrils. We probed specifically the surface of the two amyloid types with high spatial resolution using tipenhanced Raman spectroscopy (TERS). The surface of the fibrils is 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. To elucidate the origin of the difference in morphology and molecular structure, we studied the assembly kinetics using a combination of atomic force microscopy and mass spectrometry. We show that at high β-lg concentrations peptide refolding into amyloid fibrils takes place faster than at low concentrations. In summary, our data show that the two fibril types are structurally different and that it is only possible to detect differences in molecular structure using surface-specific spectroscopic techniques. The origin of the difference in molecular structure is likely related to the faster formation kinetics at high protein concentrations. Multimodal spectroscopic study of amyloid polymorphism 65