Formation of amyloid fibrils from fully reduced hen egg white lysozyme

Abstract

The fully reduced hen egg white lysozyme (HEWL), which is a good model of random coil structure, has been converted to highly organized amyloid fibrils at low pH by adding ethanol. In the presence of 90% (v/v) ethanol, the fully reduced HEWL adopts ␤-sheet secondary structure at pH 4.5 and 5.0, and an ␣-to-␤ transition is observed at pH 4.0. A red shift of the Congo red absorption spectrum caused by the precipitation of the fully reduced HEWL in the presence of 90% (v/v) ethanol is typical of the presence of amyloid aggregation. EM reveals unbranched fibrils with a diameter of 2-5 nm and as long as 1-2 m. The pH dependence of the initial structure of the fully reduced HEWL in the presence of 90% (v/v) ethanol suggests that Asp and His residues may play an important role.Keywords: Amyloid fibril formation; hen lysozyme; disulfide reduction Protein amyloid aggregation has been recognized as a major cause of several important diseases, including Alzheimer's disease (the fourth most common cause of death in the Western world), Parkinson's disease, type II or noninsulindependent diabetes, and the transmissible spongiform encephalopathies. About 17 different proteins have been found to form amyloid in vivo. Amyloid fibrils formed from those proteins share some common morphological features, but these proteins do not have a conserved sequence or native structural motif (Kelly 1996;Lansbury 1999).Recent studies show that amyloid formation is not only possible with disease-associated proteins, but also with proteins that are not associated with any known amyloid diseases under certain conditions (Guijarro et al. 1998;Chiti et al. 1999;Dobson 1999;Fandrich et al. 2001;Kallberg et al. 2001). Hen egg white lysozyme (HEWL), an extensively studied small protein with four disulfide bonds, is a recent example (Goda et al. 2000;Krebs et al. 2000). Amyloid fibrils formed from all these proteins that are not associated with any known amyloid diseases not only share similar morphological features with amyloid fibrils from diseaseassociated proteins, but also can be inherently highly cytotoxic (Bucciantini et al. 2002). Based on these observations, Dobson proposed that amyloid aggregation was a generic property of all polypeptides (Guijarro et al. 1998;Dobson 1999).Study of the amyloid aggregation of nondisease-associated proteins cannot only help us understand the mechanism of amyloid fibrillogenesis, but also extend our understanding of the basic relationship between protein sequence and structure. A common strategy to convert a nondisease-associated protein to amyloid fibrils is destabilizing the protein either by mutation or by partial denaturation with heating or addition of alcohols (Chiti et al. 1999;Rochet and Lansbury 2000). Most of the above studies are based on partially denaturing the native structure of the target proteins; in other words, those target proteins still maintain a substantial amount of their native structures. One question remaining is whether or not unstructured polypeptides can form amyloid fibri...

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

“…It has been postulated that a misfolded/partially unfolded intermediate is a prerequisite for the formation of fibrils (Bellotti et al 2000;Goers et al 2002;Cao et al 2004;Uversky and Fink 2004;Surewicz et al 2006). Irreversible partial unfolding of hen egg white lysozyme has been recently reported ) to occur at the early stages of fibrillation at elevated temperature and Figure 9.…”

“…Protein concentrations were determined by diluting the samples into 6 M guanidine solution and measuring the UV absorbance at 280 nm. A 280nm (1 mg/mL) ¼ 2.37 was used for calculating the concentration of lysozyme (Cao et al 2004). …”

“…It is thought that an intermediate, which is devoid of well-characterized tertiary structure while retaining a certain level of secondary structure, is essential for the conformational changes that lead to the fibril formation (Bellotti et al 2000;Goers et al 2002;Cao et al 2004;Uversky and Fink 2004;Surewicz et al 2006). This partially unfolded intermediate shares many conformational properties with an early intermediate of protein folding, a premolten globule state.…”

“…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 .…”

Unfolding and aggregation of lysozyme: A thermodynamic and kinetic study by FTIR spectroscopy

“…sheet structures whose stability is enhanced upon a temperature decrease (down to room temperature) [17][18][19][20][21].…”