Tip-Enhanced Raman Scattering Reveals Heterogeneity of Secondary Structures in Amyloid Fibrils Formed by Peptide CGNNQQNY

Abstract

Tip-enhanced Raman Scattering (TERS) is an advanced technique allowing to measure Raman signals with high spatial resolution. By utilizing SERS effect in a probe coupled with Scanning Probe Microscopy the resolution reaches only a few nanometers breaking diffraction limit of optical microscopy. We have used TERS to study conformational features of amyloid aggregates formed by a peptide (CGNNQQNY) from the yeast prion protein Sup35. TERS results allowed us to identify a set of conformations that composes surfaces of morphologically different amyloid fibrils. Analysis of characteristic Raman bands in the amide III region showed that fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (β-sheets, unordered and α-helix) while fibrils formed at pH 2 have primarily β-sheets. Additionally, by correlating the Raman peak positions corresponding to β-sheet structure in the amide III region with Ψ dihedral angle obtained from Molecular Dynamics simulations of 16-mer fibrils, we proposed that peptides have parallel arrangement of β-sheets for pH 2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We developed a methodology for detailed analysis of the peptide secondary structure that enabled us to correlate intensity changes in TERS spectra. Extensive cross correlation analysis of Raman peak intensities corresponding to α-helix, β-sheet and unordered secondary structures in amide III region and amide I region indicated that both regions provide adequate representation of structural characteristics of amyloid surfaces. Furthermore, 2D correlation with the band corresponding to Cα-H in plane bending suggested high localization of structural properties and large contribution of unordered non-helical secondary structures. Here we also discuss various ways to prepare TERS active probes, their longevity and enhancement capabilities suitable for protein aggregation studies.Acknowledgements: NIH-GM096039, NSF-EPS-1004094 to YLL and 2P20GM103480-07 to AVK.