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...
The early stages of hen egg white lysozyme (HEWL) fibrillation were quantitatively characterized by two-dimensional correlation deep UV resonance Raman spectroscopy (2D-DUVRR) in terms of the sequential order of events and their characteristic times. The evolution of individual secondary structural elements was established through the correlation between Amide I, Amide III, and Calpha-H bending Raman bands. The temporal order of tertiary and individual secondary structural changes was probed through the cross-correlation of phenylalanine and Amide Raman bands. Both the sequential order and the characteristic times of tertiary and secondary structural changes allowed for reconstructing the molecular mechanism of lysozyme structural changes at the early stages of fibrillation. The 2D-DUVRR analysis of our data indicated that melting of the alpha-helix happened after the formation of the disordered structure, which was termed as apparent inverse order of secondary structural changes. We demonstrated that this apparent inverse order of events is typical for all chemical reactions involving the formation of intermediate(s), which may lead to the serious misinterpretation of 2D correlation results. We proposed a new simulation-aided approach for reconstructing and quantitatively characterizing the reaction mechanism of a (bio)chemical reaction that accounts for the apparent inverse order of events.
Amyloid fibril depositions are associated with many neurodegenerative diseases. The nucleation step of fibrillation remains poorly characterized as no experimental technique allows for direct monitoring of nucleus formation. A new method based on 2D-correlation deep UV resonance Raman spectroscopy was applied to probe directly all species at early stages of lysozyme fibrillation, establish the sequential order of highly correlated events, and characterize quantitatively the kinetics of nucleus formation.
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