Polymorphism of amyloid fibrils formed by a peptide from the yeast prion protein Sup35: AFM and Tip-Enhanced Raman Scattering studies

Abstract

Aggregation of prion proteins is the cause of various prion related diseases. The infectious form of prions, amyloid fibrils, exist as multiple strains. The strains are thought to represent structurally different prion protein molecules packed into amyloid fibrils, but the knowledge on the structure of different types of aggregates is limited. Here we report on the use of AFM (Atomic Force Microscopy) and TERS (Tip-Enhanced Raman Scattering) to study morphological heterogeneity and access underlying conformational features of individual amyloid aggregates. Using AFM we identified morphology of amyloid fibrils formed by the peptide (CGNNQQNY) from the yeast prion protein Sup35 that is critically involved in the aggregation of the full protein. TERS results demonstrate that morphologically different amyloid fibrils are composed of a distinct set of conformations. Fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (β-sheets, random coil and α-helix) while fibrils formed in pH~2 solution primarily have β-sheets. Additionally, peak positions in the amide III region of the TERS spectra suggested that peptides have parallel arrangement of β-sheets for pH~2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We also developed a methodology for detailed analysis of the peptide secondary structure by correlating intensity changes of Raman bands in different regions of TERS spectra. Such correlation established that structural composition of peptides is highly localized with large contribution of unordered secondary structures on a fibrillar surface.

Tip‐Enhanced Raman Spectroscopy to Distinguish Toxic Oligomers from Aβ1–42 Fibrils at the Nanometer Scale

“…Note that the smoothness of the Au was corroborated by the absence of SERS signals of the amyloid peptides.T oprevent thermal and photochemical decomposition, al ow fluence of 0.55 nW nm À2 was used. This value is 4-14-fold lower than that reported in the literature [2,13] for the study of similar samples (experimental details in the Supporting Information). Thei ntegrity of the three specimens under irradiation was also indicated by AFM height and phase images recorded before and after light excitation.…”

“…A thorough assignment of all the expected Raman peaks for amyloid peptides is available in the literature. [2,[13][14][15][16][17][18] In practice,m ost of the vibrational modes tentatively assigned to amino acid residues have weak intensities and can be attributed to multiple residues,w hich complicates their use for toxicity discrimination. Fori nstance,t he Raman peak at 767(AE 3) cm À1 can be assigned to the C À Sstretching vibration of cysteine (C) or methionine (M).…”

“…Selected Ag/AuT ERS probes had 60 nm apex curvature radii, as confirmed by scanning electron microscopy (SEM) images of three metalized tips ( Figure S1 and experimental details in the Supporting Information). This value is 4-14-fold lower than that reported in the literature [2,13] for the study of similar samples (experimental details in the Supporting Information). Theg ap-mode configuration was carried out using as mooth 10 nm-thick Au film (with < 1nmr oughness) sputtered on top of ac lean silicate glass coverslip.Samples were immediately spin-coated on top of freshly metallized substrates to avoid contamination.…”

Fabrication of a Biocompatible Mica/Gold Surface for Tip‐Enhanced Raman Spectroscopy

“…This application of TERS has thus far been limited, due to difficulties in generating high field enhancements while maintaining biocompatibility. [4][5][6][7][8][9][10][11][12] However, there remain significant challenges to its widespread implementation and capacity to reliably address biological and clinical questions.The sensitivity of TERS arises from the excitation of a localized plasmon resonance in a noble metal AFM tip, which can provide an order of magnitude enhancement in the optical field close to the tip. However, metals have poor biocompatibility, potentially introducing difficulties in characterizing native structure and conformation in biomolecules, whereas biocompatible surfaces have weak optical field enhancements.…”

“…AFM is a powerful technique for imaging on the single-molecule level, [1] and Raman spectroscopy can distinguish biological constituents and their organization and conformation through their vibrational signatures. [4][5][6][7][8][9][10][11][12] However, there remain significant challenges to its widespread implementation and capacity to reliably address biological and clinical questions. [4][5][6][7][8][9][10][11][12] However, there remain significant challenges to its widespread implementation and capacity to reliably address biological and clinical questions.…”