title: New insight into amyloid fibril formation of hen egg white lysozyme using a two-step temperature-dependent FTIR approach.
abstract: 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.
authors: Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, College of Chemistry and Environmental Science, Hebei University , Baoding 071002, China.
authors: Ye
authors: Y
authors: Zou
authors: Wenying
authors: W
authors: Hao
authors: Haoyi
authors: H
authors: Li
authors: Yachao
authors: Y
authors: Gao
authors: Ying
authors: Y
authors: Sun
authors: Gang
authors: G
authors: Ma
mesh_terms: D000682:Amyloid / Q000737:chemistry; D000818:Animals; D002645:Chickens; D017666:Deuterium Oxide / Q000737:chemistry; D018625:Microscopy, Atomic Force; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D000682:Amyloid; C421864:hen egg lysozyme; D009113:Muramidase; D017666:Deuterium Oxide