2D Correlation Deep UV Resonance Raman Spectroscopy of Early Events of Lysozyme Fibrillation:  Kinetic Mechanism and Potential Interpretation Pitfalls

Abstract

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.

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

“…Advanced statistical methods allow for retrieving of reliable information from data sets that is not otherwise evident. We have been successful in developing and applying advanced statistical analysis of Raman spectra for biochemical [19][20][21][22][23][24][25][26][27] and forensic purposes [7][8][9]11,13].…”

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

“…We illustrate this approach by reviewing our studies of the structural rearrangements of hen egg white lysozyme at the initial stage of fibril formation. [53,54] …”

“…Since the spectra of pure secondary structures are not observable in the experiment, we proposed to use the calculated pure component spectra as the closest estimate of the basis secondary structure spectra. [53] These pure component spectra are calculated by independent component analysis (ICA), [77] pure variable [77] and Bayesian source separation approaches. [78] The proposed approach discovered the source of potential misinterpretation of 2DCoS results.…”

“…[53] we extended the 2D-DUVRR analysis of the early stage of lysozyme fibrillation from the C α -H region [54] to amide I, amide III, and phenylalanine 1000 cm −1 bands. The sequential order of secondary structure changes (α-helix, β-sheet and random coil) was determined and interpreted based on the previously proposed kinetics scheme.…”

Two-Dimensional Correlation Analysis of Sum-Frequency Vibrational Spectra of Langmuir Monolayers

“…Nowadays, a more advanced formalism based on Hilbert transformation is widely employed for calculation because of its computational efficiency and simplicity [17]. To date, 2D correlation analysis has been utilized to analyze many kinds of one-dimensional (1D) spectroscopic data including electronic, IR, NMR, X-ray and Raman spectra, and was found useful for an in-depth study of spectral variations caused by external factors [18][19][20][21][22][23][24]. Recently, Hore and coworkers first suggested applying 2D correlation analysis to investigate the simulated sum-frequency spectra, and demonstrated that 2D correlation analysis can also be readily applied to the sum-frequency vibrational spectroscopy [25].…”