Lysozyme fibrillation: Deep UV Raman spectroscopic characterization of protein structural transformation

Abstract

Deep ultraviolet resonance Raman spectroscopy was demonstrated to be a powerful tool for structural characterization of protein at all stages of fibril formation. The evolution of the protein secondary structure as well as the local environment of phenylalanine, a natural deep ultraviolet Raman marker, was documented for the fibrillation of lysozyme. Concentration-independent irreversible helix melting was quantitatively characterized as the first step of the fibrillation. The native lysozyme composed initially of 32% helix transforms monoexponentially to an unfolded intermediate with 6% helix with a characteristic time of 29 h. The local environment of phenylalanine residues changes concomitantly with the secondary structure transformation. The phenylalanine residues in lysozyme fibrils are accessible to solvent in contrast to those in the native protein.

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two‐state transition

“…The preparation of lysozyme fibrils was described elsewhere . Briefly, ;14 mg/mL of lysozyme solution with pH 2.0 was prepared and incubated at 65°C for various durations (Krebs et al 2000;Xu et al 2005). The incubated solutions were centrifuged at 16,000 g for 30 min to separate the soluble fraction from the gelatinous fraction that is dominated by fibrils.…”

“…Utilization of UV irradiation at ;200 nm enables selective resonance enhancement of vibrational modes from amide chromophore (i.e., peptide bond), the building block of a polypeptide backbone. The technique, deep UV resonance Raman (DUVRR) spectroscopy, has been shown to be a powerful tool for structural characterization of proteins and polypeptides Asher 2001;Lednev et al 2005;Xu et al 2005;JiJi et al 2006). We have recently demonstrated that DUVRR spectroscopy can be used for probing structural evolution of an amyloidogenic protein at all stages of fibril formation .…”

“…In the case of hen egg white lysozyme (herein refer to as lysozyme), conditions such as heating (Krebs et al 2000;Arnaudov and De Vries 2005;Xu et al 2005), chemical denaturation (Vernaglia et al 2004), chemical denaturation and reduction (Cao et al 2004;Gu et al 2004), addition of alcohols (Hoshino et al 1997;Goda et al 2000;Cao et al 2004), etc., have been reported to facilitate the formation of fibrils. Using DUVRR spectroscopy, we have recently shown that an irreversible unfolding of hen egg white lysozyme at 65°C and low pH is the first step of fibrillation .…”

Hen egg white lysozyme fibrillation: a deep‐UV resonance Raman spectroscopic study

“…This is because the macroscopic properties of an amyloidogenic protein solution change during the fibrils formation in vitro: a gelatinous phase appears, which is mainly composed from fibrils [59]. The insoluble phase poses a serious problem in applying conventional biophysical techniques for studying protein structural transformations during the fibril formation.…”

“…The insoluble phase poses a serious problem in applying conventional biophysical techniques for studying protein structural transformations during the fibril formation. Raman spectroscopy, by its nature, is based on inelastic light scattering [60] and can be utilized equally well for both transparent and highly light-scattering samples [59,61,62]. When the incident light interacts with a molecule, the scattered photons might lose energy due to the excitation of molecular vibrations.…”