Multimodal Spectroscopic Study of Amyloid Fibril Polymorphism

Abstract

Amyloid fibrils are a large class of self-assembled protein aggregates that are formed from unstructured peptides and unfolded proteins. The fibrils are characterized by a universal β-sheet core stabilized by 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 varying the protein concentration during formation. We investigate the differences in the molecular structure and composition between long, straight fibrils versus short, wormlike 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 dominant contribution of the β-sheet core but no difference in structure between straight and wormlike fibrils. However, when probing specifically the surface of the fibrils with nanometer resolution using tip-enhanced Raman spectroscopy (TERS), we find that both fibril types exhibit a heterogeneous surface structure with mainly unordered or α-helical structures and that the surface of long, straight fibrils contains markedly more β-sheet structure than the surface of short, wormlike fibrils. This finding is consistent with previous surface-specific vibrational sum-frequency generation (VSFG) spectroscopic results ( VandenAkker et al. J. Am. Chem. Soc. , 2011 , 133 , 18030 - 18033 , DOI: 10.1021/ja206513r ). In conclusion, only advanced vibrational spectroscopic techniques sensitive to surface structure such as TERS and VSFG are able to reveal the difference in structure that underlies the distinct morphology and rigidity of different amyloid fibril polymorphs that have been observed for a large range of food and disease-related proteins.

Tip‐Enhanced Raman Spectroscopy: A Tool for Nanoscale Chemical and Structural Characterization of Biomolecules

“…Table lists TERS wavenumbers for several secondary structures (α helix, β sheet, random coil/unordered) deduced from the thorough investigation of amide I (whenever observable) and amide III bands. TERS data, obtained with a spatial resolution down to 2 nm or less, suggest a difference in secondary structure between the core and apparently very heterogeneous surface of amyloid fibers . A chemical distinction between the surface of mature fibrils and that of protofilaments, which are their precursors, was also demonstrated by using TERS .…”

“…Cys is associated with two bands assigned to C−S stretches, in the ranges 806–745 and 698–650 cm −1 . The Fermi resonance doublet of Tyr is easily recognizable in the 868–819 cm −1 spectral region . The NH indole ring stretching mode constitutes a clear fingerprint of Trp in the range 1455–1423 cm −1 .…”

“…The NH indole ring stretching mode constitutes a clear fingerprint of Trp in the range 1455–1423 cm −1 . Two intense and sharp bands reveal the presence of Phe residues in proteins primary sequences: one in the range 1014–997 cm −1 and the other in the range 1056–1018 cm −1 . In the case of His, if a combination of three TERS bands centered at 1180, 1330, and 1495 cm −1 appears in TERS spectra, it can be used as a His spectral marker, although the corresponding vibrational modes are not individually specific to His .…”

“…The TERS technique was used to study the conformational changes in the secondary structure of self‐assembled peptide and the distribution of the (hydrophilic or hydrophobic) amino acids on the surface of the fibers formed from various proteins, such as insulin, human islet amyloid polypeptide (HIAPP), β‐lactoglobulin, or peptide from Sup35 prion proteins . The aromatic amino acids (Phe, Trp, and Tyr), Cys, His, and Pro were predominantly detected . However, the TERS footprints of Arg, Lys, Asn, and Gln sometimes appeared .…”

Binuclear ruthenium complexes inhibit the fibril formation of human islet amyloid polypeptide

“…3-(4,5-Dimethyl-2-thiazolyl)-2,5diphenyltetrazolium bromide (MTT) used to detect the toxicity of amyloid peptide was purchased from Sigma. 38 All other reagents were of analytical grade.…”