Probing the Cross-β Core Structure of Amyloid Fibrils by Hydrogen−Deuterium Exchange Deep Ultraviolet Resonance Raman Spectroscopy

Abstract

Studying the structure of amyloid fibrils is important for the detailed understanding of fibrillogenesis at a molecular level. Amyloid fibrils are noncrystalline and insoluble, and thus are not amenable to conventional X-ray crystallography and solution NMR, the classical tools of structural biology. 1 Several specialized techniques with less general capabilities have been developed and utilized for probing fibrillar structure. Transmission electron microscopy (TEM) and scanning probe microscopy (SPM) provide general information on fibril topology and morphology including the length, width, interstrand spacing, the number of protofilaments, and the way they are assembled to form the fibril. 1,2 The application of fiber X-ray diffraction and scattering has been limited to short peptides mimicking the core structure of the fibrils formed from amyloidogenic protein. [3][4][5] Wide-angle X-ray scattering from floworiented fibrils has been utilized to estimate interstrand and intersheet spacing in cross-structures. 6 Solid-state NMR probes interatomic distances and torsion angles, which define local secondary structure and side-chain conformations. This technique, however, requires site-specific 13 C and/or 15 N labels. 7 Further, FTIR combined with proteolysis has been used to characterize the core structure of lysozyme fibrils. 8 Application of FTIR, however, is limited because of intense IR absorption by water. Here we report on the first application of hydrogen-deuterium exchange (HX) deep UV resonance Raman (DUVRR) spectroscopy to probe the secondary structure of the fibril cross-core. The amide I bandwidth in the DUVRR spectrum of the highly ordered cross-sheet was found comparable to that of the Gly-Gly crystal, indicating no inhomogeneous broadening due to various amino acid residues involved into the cross-core. This is in contrast to the Raman spectra of native globular protein -sheets which exhibit broader Raman peaks than those of homopolypeptides. 9,10 The dominating spectral contribution was assigned to the antiparallel -sheet. Thus, HX-DUVRR spectroscopy is a powerful tool for structural characterization of cross-core of amyloid fibrils.HX is a valuable tool for characterizing protein structure, solvation, and water exposure, when combined with NMR, 11 mass spectrometry, 12 and vibrational spectroscopy. 13 In an amino acid residue, the main chain NH group and O, N, and S bound protons exchange easily, whereas carbon-bound hydrogens do not. 14 In the protein hydrophobic core or strongly hydrogen-bonded secondary structures, the HX rates are strongly reduced owing to shielding of exchangeable sites. 13 We hypothesized that amide N-H protons in unordered fragments of amyloid fibrils should exchange readily, whereas those hidden from water in the cross-structure will remain protonated. As shown by Mikhonin and Asher, 15 HX causes a downshift of the amide II DUVRR band from ∼1555 to ∼1450 cm -1 (amide II′) and the virtual disappearance of the amide III band in an unordered protein. Figure 1a illustrates corr...

Structural differences between amyloid beta oligomers

“…The sensitivity of the amide M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 8 chromophore Raman signature to Ψ dihedral angle makes this technique uniquely capable of differentiating between globular and fibrillar β-sheet conformations and between parallel and antiparallel β-sheets [19,30]. The DUVRR spectrum of PFOs (Fig.…”

Discriminant Analysis of Raman Spectra for Body Fluid Identification for Forensic Purposes

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Two‐dimensional correlation Raman spectroscopy for characterizing protein structure and dynamics

“…Our studies have shown that amide I band of native lysozyme [77] is centered at 1660 cm −1 , whereas amide I bands of unordered lysozyme and lysozyme fibrillar β-sheet have maxima around 1667 cm −1 and 1674 cm −1 , respectively. [79] Accordingly, the high wavenumber shift of the amide I band indicates an increase in random coil or/and β-sheet contents of over the course of incubation. The noticeable sharpening of the amide I band at long incubation times is due to the formation of nucleus β-sheet structure.…”