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See detailDisease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster
Kumita, Janet R.; Helmfors, Linda; Williams, Jocy et al

in FASEB Journal (2012)

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic ... [more ▼]

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.-Kumita, J. R., Helmfors, L., Williams, J., Luheshi, L. M., Menzer, L., Dumoulin, M., Lomas, D. A., Crowther, D. C., Dobson, C. M., Brorsson, A.-C. Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster. [less ▲]

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See detailPopulation of nonnative States of lysozyme variants drives amyloid fibril formation.
Buell, Alexander K.; Dhulesia, Anne; Mossuto, Maria F. et al

in Journal of the American Chemical Society (2011), 133(20), 7737-43

The propensity of protein molecules to self-assemble into highly ordered, fibrillar aggregates lies at the heart of understanding many disorders ranging from Alzheimer's disease to systemic lysozyme ... [more ▼]

The propensity of protein molecules to self-assemble into highly ordered, fibrillar aggregates lies at the heart of understanding many disorders ranging from Alzheimer's disease to systemic lysozyme amyloidosis. In this paper we use highly accurate kinetic measurements of amyloid fibril growth in combination with spectroscopic tools to quantify the effect of modifications in solution conditions and in the amino acid sequence of human lysozyme on its propensity to form amyloid fibrils under acidic conditions. We elucidate and quantify the correlation between the rate of amyloid growth and the population of nonnative states, and we show that changes in amyloidogenicity are almost entirely due to alterations in the stability of the native state, while other regions of the global free-energy surface remain largely unmodified. These results provide insight into the complex dynamics of a macromolecule on a multidimensional energy landscape and point the way for a better understanding of amyloid diseases. [less ▲]

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See detailStructure and properties of a complex of alpha-synuclein and a single-domain camelid antibody.
De Genst, Erwin J; Guilliams, Tim; Wellens, Joke et al

in Journal of Molecular Biology (2010), 402(2), 326-43

The aggregation of the intrinsically disordered protein alpha-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's ... [more ▼]

The aggregation of the intrinsically disordered protein alpha-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's disease. The molecular mechanism of alpha-synuclein aggregation and toxicity is not yet understood in any detail, not least because of the paucity of structural probes through which to study the behavior of such a disordered system. Here, we describe an investigation involving a single-domain camelid antibody, NbSyn2, selected by phage display techniques to bind to alpha-synuclein, including the exploration of its effects on the in vitro aggregation of the protein under a variety of conditions. We show using isothermal calorimetric methods that NbSyn2 binds specifically to monomeric alpha-synuclein with nanomolar affinity and by means of NMR spectroscopy that it interacts with the four C-terminal residues of the protein. This latter finding is confirmed by the determination of a crystal structure of NbSyn2 bound to a peptide encompassing the nine C-terminal residues of alpha-synuclein. The NbSyn2:alpha-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of alpha-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of alpha-synuclein at physiological pH, including the morphology of the resulting fibrillar structures, is remarkably unaffected by the presence of NbSyn2 and indeed we show that NbSyn2 binds strongly to the aggregated as well as to the soluble forms of alpha-synuclein. These results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of alpha-synuclein aggregation in vitro and possibly in vivo. [less ▲]

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See detailThe non-core regions of human lysozyme amyloid fibrils influence cytotoxicity.
Mossuto, Maria F; Dhulesia, Anne; Devlin, Glyn et al

in Journal of Molecular Biology (2010), 402(5), 783-96

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of ... [more ▼]

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-beta core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties. [less ▲]

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See detailA non-natural variant of human lysozyme (I59T) mimics the in vitro behaviour of the I56T variant that is responsible for a form of familial amyloidosis.
Hagan, Christine L; Johnson, Russell J K; Dhulesia, Anne et al

in Protein Engineering, Design & Selection (2010), 23(7), 499-506

We report here the detailed characterisation of a non-naturally occurring variant of human lysozyme, I59T, which possesses a destabilising point mutation at the interface of the alpha- and beta-domains ... [more ▼]

We report here the detailed characterisation of a non-naturally occurring variant of human lysozyme, I59T, which possesses a destabilising point mutation at the interface of the alpha- and beta-domains. Although more stable in its native structure than the naturally occurring variants that give rise to a familial form of systemic amyloidosis, I59T possesses many attributes that are similar to these disease-associated species. In particular, under physiologically relevant conditions, I59T populates transiently an intermediate in which a region of the structure unfolds cooperatively; this loss of global cooperativity has been suggested to be a critical feature underlying the amyloidogenic nature of the disease-associated lysozyme variants. In the present study, we have utilised this variant to provide direct evidence for the generic nature of the conformational transition that precedes the ready formation of the fibrils responsible for lysozyme-associated amyloid disease. This non-natural variant can be expressed at higher levels than the natural amyloidogenic variants, enabling, for example, singly isotopically labelled protein to be generated much more easily for detailed structural studies by multidimensional NMR spectroscopy. Moreover, we demonstrate that the I59T variant can readily form fibrils in vitro, similar in nature to those of the amyloidogenic I56T variant, under significantly milder conditions than are needed for the wild-type protein. [less ▲]

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See detailCharacterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme
Frare, Erica; Mossuto, Maria F.; Polverino de Laureto, Patrizia et al

in Journal of Molecular Biology (2009)

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See detail(1)H, (13)C and (15)N assignments of a camelid nanobody directed against human alpha-synuclein.
Vuchelen, Anneleen; O'Day, Elizabeth; De Genst, Erwin et al

in Biomolecular NMR Assignments (2009), 3(2), 231-3

Nanobodies are single chain antibodies that are uniquely produced in Camelidae, e.g. camels and llamas. They have the desirable features of small sizes (Mw < 14 kDa) and high affinities against antigens ... [more ▼]

Nanobodies are single chain antibodies that are uniquely produced in Camelidae, e.g. camels and llamas. They have the desirable features of small sizes (Mw < 14 kDa) and high affinities against antigens (Kd approximately nM), making them ideal as structural probes for biomedically relevant motifs both in vitro and in vivo. We have previously shown that nanobody binding to amyloidogenic human lysozyme variants can effectively inhibit their aggregation, the process that is at the origin of systemic amyloid disease. Here we report the NMR assignments of a new nanobody, termed NbSyn2, which recognises the C-terminus of the intrinsically disordered protein, human alpha-synuclein (aS), whose aberrant self-association is implicated in Parkinson's disease. [less ▲]

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See detailCharacterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme
Frare, Erica; Mossuto, Maria F.; Polverino de Laureto, Patrizia et al

in Journal of Molecular Biology (2009), 387

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 ... [more ▼]

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 transforminfrared 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 transforminfrared 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. [less ▲]

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See detailOptimization of the Production of the Amyloidogenic Variants of Human Lysozyme
Menzer, Linda ULg; Tocquin, Pierre ULg; Dony, Nicolas et al

Poster (2008, February 16)

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See detailEngineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils
Chan, Pak Ho; Pardon, Els; Menzer, Linda ULg et al

in Biochemistry (2008), 47

single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic ... [more ▼]

single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic variants of human lysozyme, which are associated with a form of systemic amyloidosis, have been investigated by a wide range of biophysical techniques. Pulse-labeling hydrogen-deuterium exchange experiments monitored by mass spectrometry reveal that binding of the antibody fragment strongly inhibits the locally cooperative unfolding of the I56T and D67H variants and restores their global cooperativity to that characteristic of the wild-type protein. The antibody fragment was, however, not stable enough under the conditions used to explore its ability to perturb the aggregation behavior of the lysozyme amyloidogenic variants. We therefore engineered a more stable version of cAb-HuL22 by adding a disulfide bridge between the two beta-sheets in the hydrophobic core of the protein. The binding of this engineered antibody fragment to the amyloidogenic variants of lysozyme inhibited their aggregation into fibrils. These findings support the premise that the reduction in global cooperativity caused by the pathogenic mutations in the lysozyme gene is the determining feature underlying their amyloidogenicity. These observations indicate further that molecular targeting of enzyme active sites, and of protein binding sites in general, is an effective strategy for inhibiting or preventing the aberrant self-assembly process that is often a consequence of protein mutation and the origin of pathogenicity. Moreover, this work further demonstrates the unique properties of camelid single-domain antibody fragments as structural probes for studying the mechanism of aggregation and as potential inhibitors of fibril formation. [less ▲]

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See detailThe extracellular chaperone clusterin potently inhibits human lysozyme amyloid formation by interacting with prefibrillar species
Kumita, Janet R.; Poon, Stephen; Caddy, Gemma L. et al

in Journal of Molecular Biology (2007), 369

We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid ... [more ▼]

We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid disease. The aggregation of the amyloidogenic variant I56T is inhibited significantly at clusterin to lysozyme ratios as low as 1:80 (i.e. one clusterin molecule per 80 lysozyme molecules). Experiments indicate that under the conditions where inhibition of aggregation occurs, clusterin does not bind detectably to the native or fibrillar states of lysozyme, or to the monomeric transient intermediate known to be a key species in the aggregation reaction. Rather, it seems to interact with oligomeric species that are present at low concentrations during the lag (nucleation) phase of the aggregation reaction. This behavior suggests that clusterin, and perhaps other extracellular chaperones, could have a key role in curtailing the potentially pathogenic effects of the misfolding and aggregation of proteins that, like lysozyme, are secreted into the extracellular environment. [less ▲]

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See detailImpact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris
Kumita, Janet; Johnson, Russel; Alcocer, Marcos et al

in FEBS Journal (2006), 273

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods ... [more ▼]

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild-type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200- and 30-fold lower, respectively, than that of wild-type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins, under controlled conditions, can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post-transcriptional processes, and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly, our results show that the human disease-associated mutations do not have levels of expression that are out of line with destabilizing mutations at other sites. These findings indicate that a complex interplay between reduced native-state stability, lower secretion levels, and protein aggregation propensity influences the types of mutation that give rise to familial forms of amyloid disease. [less ▲]

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See detailNormal and aberrant biological self-assembly: Insights from studies of human lysozyme and its amyloidogenic variants
Dumoulin, Mireille ULg; Kumita, Janet; Dobson, Christopher M

in Accounts of Chemical Research (2006), 39

Studies of lysozyme have played a major role over several decades in defining the general principles underlying protein structure, folding, and stability. Following the discovery some 10 years ago that ... [more ▼]

Studies of lysozyme have played a major role over several decades in defining the general principles underlying protein structure, folding, and stability. Following the discovery some 10 years ago that two mutational variants of lysozyme are associated with systemic amyloidosis, these studies have been extended to investigate the mechanism of amyloid fibril formation. This Account describes our present knowledge of lysozyme folding and misfolding, and how the latter can give rise to amyloid disease. It also discusses the significance of these studies for our general understanding of normal and aberrant protein folding in the context of human health and disease. [less ▲]

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See detailIdentification of the Core Structure of Lysozyme Amyloid Fibrils by Proteolysis
Frare, Erica; Mossuto, Maria F.; Polverino de Laureto, Patrizia et al

in Journal of Molecular Biology (2006), 361

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 ... [more ▼]

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. [less ▲]

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See detailReduced global copperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations
Dumoulin, Mireille ULg; Canet, Denis; Last, Alexander M. et al

in Journal of Molecular Biology (2005), 346(3), 773-788

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data ... [more ▼]

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data that permit a detailed comparison to be made of the behaviour of two of these amyloidogenic variants, I56T and D67H, under identical conditions. Hydrogen/deuterium exchange experiments monitored by NMR and mass spectrometry reveal that, despite their different locations and the different effects of the two mutations on the structure of the native state of lysozyme, both mutations cause a cooperative destabilisation of a remarkably similar segment of the structure, comprising in both cases the beta-domain and the adjacent C-helix. As a result, both variant proteins populate transiently a closely similar, partially unstructured intermediate in which the beta-domain and the adjacent C-helix are substantially and simultaneously unfolded, whereas the three remaining a-helices that form the core of the a-domain still have their native-like structure. We show, in addition, that the binding of a camel antibody fragment, cAb-HuL6, which was raised against wild-type lysozyme, restores to both variant proteins the stability and cooperativity characteristic of the wild-type protein; as a consequence, it inhibits the formation of amyloid fibrils by both variants. These results indicate that the reduction in global cooperativity, an associated ability to populate transiently a specific, partly unfolded intermediate state under physiologically relevant conditions, is a common feature underlying the behaviour of these two pathogenic mutations. The formation of intermolecular interactions between lysozyme molecules that are in this partially unfolded state is therefore likely to be the fundamental trigger of the aggregation process that ultimately leads to the formation and deposition in tissue of amyloid fibrils. (C) 2004 Elsevier Ltd. All rights reserved. [less ▲]

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See detailRationalising Lysozyme Amyloidosis: Insights from the Structure and Solution Dynamics of T70N Lysozyme
Johnson, Russell J.K.; Christodoulou, John; Dumoulin, Mireille ULg et al

in Journal of Molecular Biology (2005), 352

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with ... [more ▼]

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45–51 and 68–75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5 Å. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70–74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the β-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the α-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the β-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo. [less ▲]

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See detailThe Folding Process of Hen Lysozyme: A Perspective from the 'New View'
Matagne, André ULg; Dobson, Christopher M.

in Cellular and Molecular Life Sciences : CMLS (1998), 54(4), 363-71

How a conformationally disordered polypeptide chain rapidly and efficiently achieves its well-defined native structure is still a major question in modern structural biology. Although much progress has ... [more ▼]

How a conformationally disordered polypeptide chain rapidly and efficiently achieves its well-defined native structure is still a major question in modern structural biology. Although much progress has been made towards rationalizing the principles of protein structure and dynamics, the mechanism of the folding process and the determinants of the final fold are not yet known in any detail. One protein for which folding has been studied in great detail by a combination of diverse techniques is hen lysozyme. In this article we review the present state of our knowledge of the folding process of this enzyme and focus in particular on recent experiments to probe some of its specific features. These results are then discussed in the context of the 'new view' of protein folding based on energy surfaces and landscapes. It is shown that a schematic energy surface for lysozyme folding, which is broadly consistent with our experimental data, begins to provide a unified model for protein folding through which experimental and theoretical ideas can be brought together. [less ▲]

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