Hen egg white lysozyme (HEWL) is widely used as a model protein for amyloid research. In this study, we aim to use Fourier transform infrared (FTIR) spectroscopy to gain new structural insights into amyloid formation of HEWL under heat and acidic condition. We reveal that the fibril-forming solution of HEWL has the capability to form fibril and oligomer with distinct β-sheet configurations under different temperatures. Amyloid fibril with parallel β-sheet configuration is formed at elevated temperature, while oligomer with antiparallel β-sheet configuration is formed at room temperature. The interplay between fibrillation and oligomerization suggests that the two β-sheet aggregates consume the same amyloidogenic materials such as peptide fragments and nicked HEWL due to lysozyme hydrolysis under heat and acidic condition. Temperature-dependent FTIR reveals that the oligomer is unstable at elevated temperature, demonstrating its off-pathway nature. The temperature-dependent formation of parallel and antiparallel β-sheet configurations discovered in lysozyme system is compared with that of amyloid-β and α-synuclein systems and the implication is discussed.
Hen egg white lysozyme (HEWL) is widely used in the mechanistic study of amyloid fibril formation. Yet, the fibrillation mechanism of HEWL is not well understood. In particular, in situ structural evidence for the on-pathway oligomeric intermediate has never been captured. Such evidence is crucial for confirming nucleated conformational conversion mechanism. Herein, we attempt to use a two-step temperature-dependent Fourier transform infrared (FTIR) approach to capture the in situ evidence for the on-pathway oligomeric intermediate and the oligomer-to-fibril transition during HEWL fibrillation. Key features of this approach include using lower temperature to generate the on-pathway oligomeric intermediate, using elevated temperature to eliminate the interference from the off-pathway oligomer and to facilitate the oligomer-to-fibril transition, and using FTIR difference spectroscopy and atomic force microscopy to tackle structure and morphology. Using such an approach, we reveal that the on-pathway oligomeric intermediate is in parallel β-sheet configuration featuring a frequency at 1622 cm(-1) and the oligomer-to-fibril transition is accompanied by a spectral transition from 1622 to 1618 cm(-1). We also discover the beneficial role of the off-pathway oligomer in the capturing of the transient on-pathway oligomeric intermediate by serving as a monomer-releasing reservoir. This approach should also be useful in other amyloidogenic systems.
Riboflavin, a common nutrient also known as vitamin B2, is known to potentially play important roles in preventing lipid peroxidations. However, the detailed antioxidant mechanisms, especially the influence of riboflavin on lipid oxidations at biological interfaces, have not yet been fully explored. In the current study, the effect of riboflavin molecules on the oxidation kinetics of monounsaturated cis-11-eicosenoic acid (EA) at the air/water interface was systematically investigated using sum frequency generation vibrational spectroscopy (SFG-VS). It was discovered that the oxidation rates of the interfacial EA molecules can be reduced by about two to three times in the presence of riboflavin in the aqueous subphase. Further SFG-VS measurements under the protection of nitrogen purging gas showed that more tightly packed and ordered monolayer structures were formed by the surface adsorption of riboflavin molecules, making the C[double bond, length as m-dash]C bonds less accessible to the gas phase oxidative species. These results suggested that the antioxidant mechanism for riboflavin in the vicinity of biomembranes may not necessarily involve other reducing agents. They also show the great importance of interfacial molecular structures in biologically relevant chemical reactions.
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