An Equilibrium Partially Folded State of Human Lysozyme at Low pH

Haezebrouck, Petra; Joniau, Marcel; Dael, H. Van; Hooke, Shaun D.; Woodruff, Nicholas D.; Dobson, Christopher M.

doi:10.1006/jmbi.1994.0093

Cited by 74 publications

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“…1). At pH 3.0, the thermal unfolding follows an apparent two-state process, as already reported, 19 with a temperature of midtransition (T m ) of 79°C. The unfolding process of lysozyme was also followed by monitoring the fluorescence emission at 475 nm of ANS, a dye whose wavelength of maximum emission shifts from 514 to 475 nm upon binding to exposed hydrophobic patches.…”

Section: 3supporting

confidence: 78%

“…Interestingly, the proteolysis of lysozyme in its oligomeric form leads to the formation of the same peptic fragments as it does in the monomeric, partially unfolded state. 19,22 Remarkably, however, within the oligomers, lysozyme is digested to a greater extent if compared with the monomeric state; after 30 min of incubation, no intact protein is evident in the proteolytic mixture, and most importantly, the three peptide bonds Asp53-Tyr54, Leu84-Leu85, and Ala92-Val93, which are located in the C-helix and the β-domain, are also cleaved ( Fig. 4a and b), while for the monomeric protein, the degree of degradation at these peptide bonds is negligible.…”

Section: Characterization Of Lysozyme Oligomersmentioning

confidence: 99%

“…14 The segment 32-108 corresponds to the region found to undergo cooperative local unfolding in the amyloidogenic variants I56T and D67H of human lysozyme 15,16 and to be amyloidogenic in the homologous hen lysozyme. 17,18 Given the high stability of wild-type human lysozyme, [19][20][21] conditions such as low pH and high temperature or ethanol have been previously used to populate a partially unfolded state that is prone to form fibrils. 14,22,23 However, these conditions known so far for the efficient formation of amyloid fibrils by human lysozyme were found not to be appropriate for this work because they either induce the fragmentation of the protein 14,18 or lead to an aggregation process that is too fast to allow isolation of oligomeric species.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

Frare

Mossuto

Laureto

et al. 2009

Journal of Molecular Biology

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The aggregation process of wild-type human lysozyme at pH 3.0 and 60°C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8-17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solventexposed hydrophobic patches able to bind the fluorescent probe 1-anilinonaphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the β-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes.

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Section: 3supporting

confidence: 78%

Section: Characterization Of Lysozyme Oligomersmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

Frare

Mossuto

Laureto

et al. 2009

Journal of Molecular Biology

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“…The experimentally-determined temperature dependence of the stability of an amyloidogenic intermediate state of lysozyme (inset of Fig. 3) [26] closely resembles the competition between stable, metastable and extended states shown in Fig. 3, thus linking our results to the misfolding process of proteins, which is often accompanied by pathogenic aggregation [26,27].…”

supporting

confidence: 73%

Importance of Metastable States in the Free Energy Landscapes of Polypeptide Chains

Auer¹,

Miller²,

Krivov

et al. 2007

Phys. Rev. Lett.

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We show that the interplay between excluded volume effects, hydrophobicity, and hydrogen bonding of a tube-like representation of a polypeptide chain gives rise to free energy landscapes that exhibit a small number of metastable minima corresponding to common structural motifs observed in proteins. The complexity of the landscape increases only moderately with the length of the chain. Analysis of the temperature dependence of these landscapes reveals that the stability of specific metastable states is maximal at a temperature close to the mid-point of folding. These mestastable states are therefore likely to be of particular significance in determining the generic tendency of proteins to aggregate into potentially pathogenic agents.PACS numbers: 87.15.Aa, 87.14.EeThe mechanism by which proteins fold reliably into their native states is frequently described by using the concept of a free energy landscape (FEL) [1,2,3,4]. Under physiological conditions, the region of configuration space associated with the native state has the lowest free energy and is therefore thermodynamically the most stable. In addition to the native and fully unfolded states, intermediate structures have been detected in the folding and misfolding processes of many proteins [5,6,7,8]. These metastable states can play an important role in the folding process, or be kinetic traps that interfere with correct folding. The experimental characterization of these states remains challenging, as they are often transient or disordered, but significant progress in this direction has recently been made by combining experiment with theory [8,9,10]. Since specific metastable states can increase the probability of misfolding and aggregation [11,12], it is important to understand the mechanism of their formation. Indeed, a global characterization of the FEL is crucial to a full understanding of the relationship between the native and metastable states, and the interplay between folding and misfolding.A complete characterization of the FEL by computational methods requires that the free energies of the native and all non-native structures be calculated. Two problems arise in this exercise: the technical issue of sampling a vast number of configurations separated by a variety of free energy barriers, and the conceptual difficulty of choosing an appropriate set of coordinates to describe the FEL [13]. Sampling is typically performed in the vicinity of an ensemble of folding or unfolding pathways, and the resulting free energy is then projected on to a subspace defined by one or two order parameters [3]. For the resulting surfaces to provide insight into thermodynamics and dynamics, it is crucial that the chosen order parameters are able to detect the relevant details of the FEL. In cases where a long dynamic trajectory that explores a large region of configuration space is available, it is possible to mitigate the order parameter problem by using the dynamics to cluster the configurations and disconnectivity graphs to represent the results [14]. To maintain th...

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“…The process was carried out at pD 1.5, 45°C, where CD data suggest that a partially unfolded state of human lysozyme is significantly populated at equilibrium (∼35%), while about 60% of the protein is still native, and the remaining 5% is highly unfolded. 67 The protein aggregation process is accelerated greatly by stirring the protein solution, so that relatively homogeneous fibrils are formed and Asp-X fragmentations occurring at low pH are avoided. 29 The only fragment found in the fibrils along with the full-length protein, before pepsin treatment, corresponds to the sequence 50-102, present at low levels, that includes a part of the β-domain and helix C of the native protein (see Figure 3(d)).…”

Section: Discussionmentioning

confidence: 99%

Identification of the Core Structure of Lysozyme Amyloid Fibrils by Proteolysis

Frare

Mossuto

Laureto

et al. 2006

Journal of Molecular Biology

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Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fouriertransform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive β-sheet structure and a substantial element of non β-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32-108 includes the β-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions.

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An Equilibrium Partially Folded State of Human Lysozyme at Low pH

Cited by 74 publications

References 0 publications

Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

Importance of Metastable States in the Free Energy Landscapes of Polypeptide Chains

Identification of the Core Structure of Lysozyme Amyloid Fibrils by Proteolysis

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