Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism.

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-ß (Aß) in Alzheimer's disease and α-synuclein (αS) in Parkinson's disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aß and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.

The Toxicity of Misfolded Protein Oligomers Is Independent of Their Secondary Structure.

The self-assembly of proteins into structured fibrillar aggregates is associated with a range of neurodegenerative diseases, including Alzheimer's and Parkinson's diseases, in which an important cytotoxic role is thought to be played by small soluble oligomers accumulating during the aggregation process or released by mature fibrils. As the structural characteristics of such species and their links with toxicity are still not fully defined, we have compared six examples of preformed misfolded protein oligomers with different ß-sheet content, as determined using Fourier transform infrared spectroscopy, and with different toxicity, as determined by three cellular readouts of cell viability. The results show the absence of any measurable correlation between the nature of their secondary structure and their cellular toxicity, both when comparing the six types of oligomers as a group and when comparing species in subgroups characterized by either the same size or the same exposure of hydrophobic moieties.

Structural differences between toxic and nontoxic HypF-N oligomers.

We have studied two misfolded oligomeric forms of the protein HypF-N, which show similar morphologies but very different toxicities. We measured over 80 intermolecular distance-dependent parameters for each oligomer type using FRET, in conjunction with solution- and solid-state NMR and other biophysical techniques. The results indicate that the formation of a highly organised hydrogen bonded core in the toxic oligomers results in the exposure of a larger number of hydrophobic residues than in the nontoxic species, causing the former to form aberrant interactions with cellular components.

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis.

Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H2). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN)2CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN)2CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase.

Crystal Structure of Carboxyltransferase from Staphylococcus aureus Bound to the Antibacterial Agent Moiramide B.

The dramatic increase in the prevalence of antibiotic-resistant bacteria has necessitated a search for new antibacterial agents against novel targets. Moiramide B is a natural product, broad-spectrum antibiotic that inhibits the carboxyltransferase component of acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid synthesis. Herein, we report the 2.6 Å resolution crystal structure of moiramide B bound to carboxyltransferase. An unanticipated but significant finding was that moiramide B bound as the enol/enolate. Crystallographic studies demonstrate that the (4S)-methyl succinimide moiety interacts with the oxyanion holes of the enzyme, supporting the notion that an anionic enolate is the active form of the antibacterial agent. Structure-activity studies demonstrate that the unsaturated fatty acid tail of moiramide B is needed only for entry into the bacterial cell. These results will allow the design of new antibacterial agents against the bacterial form of carboxyltransferase.

Interaction of toxic and non-toxic HypF-N oligomers with lipid bilayers investigated at high resolution with atomic force microscopy.

Protein misfolded oligomers are considered the most toxic species amongst those formed in the process of amyloid formation and the molecular basis of their toxicity, although not completely understood, is thought to originate from the interaction with the cellular membrane. Here, we sought to highlight the molecular determinants of oligomer-membrane interaction by atomic force microscopy. We monitored the interaction between multiphase supported lipid bilayers and two types of HypF-N oligomers displaying different structural features and cytotoxicities. By our approach we imaged with unprecedented resolution the ordered and disordered lipid phases of the bilayer and different oligomer structures interacting with either phase. We identified the oligomers and lipids responsible for toxicity and, more generally, we established the importance of the membrane lipid component in mediating oligomer toxicity. Our findings support the importance of GM1 ganglioside in mediating the oligomer-bilayer interaction and support a mechanism of oligomer cytotoxicity involving bilayer destabilization by globular oligomers within GM1-rich ordered raft regions rather than by annular oligomers in the surrounding disordered membrane domains.

Hybrid Structure of a Dynamic Single-Chain Carboxylase from Deinococcus radiodurans.

Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes.

Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate Carboxylase Facilitates Product Release from the Biotin Carboxylase Domain through an Intermolecular Mechanism.

Protein structure, ligand binding, and catalytic turnover contributes to the governance of catalytic events occurring at spatially distinct domains in multifunctional enzymes. Coordination of these catalytic events partially rests on the ability of spatially discrete active sites to communicate with other allosteric and active sites on the same polypeptide chain (intramolecular) or on different polypeptide chains (intermolecular) within the holoenzyme. Often, communication results in long-range effects on substrate binding or product release. For example, pyruvate binding to the carboxyl transferase (CT) domain of pyruvate carboxylase (PC) increases the rate of product release in the biotin carboxylase (BC) domain. In order to address how CT domain ligand occupancy is "sensed" by other domains, we generated functional, mixed hybrid tetramers using the E218A (inactive BC domain) and T882S (low pyruvate binding, low activity) mutant forms of PC. The apparent Ka pyruvate for the pyruvate-stimulated release of Pi catalyzed by the T882S:E218A[1:1] hybrid tetramer was comparable to the wild-type enzyme and nearly 10-fold lower than that for the T882S homotetramer. In addition, the ratio of the rates of oxaloacetate formation to Pi release for the WT:T882S[1:1] and E218A:T882S[1:1] hybrid tetramer-catalyzed reactions was 0.5 and 0.6, respectively, while the T882S homotetramer exhibited a near 1:1 coupling of the two domains, suggesting that the mechanisms coordinating catalytic events is more complicated that we initially assumed. The results presented here are consistent with an intermolecular communication mechanism, where pyruvate binding to the CT domain is "sensed" by domains on a different polypeptide chain within the tetramer.

Regulation and structure of the heteromeric acetyl-CoA carboxylase.

The enzyme acetyl-CoA carboxylase (ACCase) catalyzes the committed step of the de novo fatty acid biosynthesis (FAS) pathway by converting acetyl-CoA to malonyl-CoA. Two forms of ACCase exist in nature, a homomeric and heteromic form. The heteromeric form of this enzyme requires four different subunits for activity: biotin carboxylase; biotin carboxyl carrier protein; and α- and ß-carboxyltransferases. Heteromeric ACCases (htACCase) can be found in prokaryotes and the plastids of most plants. The plant htACCase is regulated by diverse mechanisms reflected by the biochemical and genetic complexity of this multienzyme complex and the plastid stroma where it resides. In this review we summarize the regulation of the plant htACCase and also describe the structural characteristics of this complex from both prokaryotes and plants. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations.

Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 µm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules.

From Genome to Structure and Back Again: A Family Portrait of the Transcarbamylases.

Enzymes in the transcarbamylase family catalyze the transfer of a carbamyl group from carbamyl phosphate (CP) to an amino group of a second substrate. The two best-characterized members, aspartate transcarbamylase (ATCase) and ornithine transcarbamylase (OTCase), are present in most organisms from bacteria to humans. Recently, structures of four new transcarbamylase members, N-acetyl-L-ornithine transcarbamylase (AOTCase), N-succinyl-L-ornithine transcarbamylase (SOTCase), ygeW encoded transcarbamylase (YTCase) and putrescine transcarbamylase (PTCase) have also been determined. Crystal structures of these enzymes have shown that they have a common overall fold with a trimer as their basic biological unit. The monomer structures share a common CP binding site in their N-terminal domain, but have different second substrate binding sites in their C-terminal domain. The discovery of three new transcarbamylases, l-2,3-diaminopropionate transcarbamylase (DPTCase), l-2,4-diaminobutyrate transcarbamylase (DBTCase) and ureidoglycine transcarbamylase (UGTCase), demonstrates that our knowledge and understanding of the spectrum of the transcarbamylase family is still incomplete. In this review, we summarize studies on the structures and function of transcarbamylases demonstrating how structural information helps to define biological function and how small structural differences govern enzyme specificity. Such information is important for correctly annotating transcarbamylase sequences in the genome databases and for identifying new members of the transcarbamylase family.

Toxicity of protein oligomers is rationalized by a function combining size and surface hydrophobicity.

The misfolding and aberrant assembly of peptides and proteins into fibrillar aggregates is the hallmark of many pathologies. Fibril formation is accompanied by oligomeric species thought to be the primary pathogenic agents in many of these diseases. With the aim of identifying the structural determinants responsible for the toxicity of misfolded oligomers, we created 12 oligomeric variants from the N-terminal domain of the E. coli HypF protein (HypF-N) by replacing one or more charged amino acid residues with neutral apolar residues and allowing the mutated proteins to aggregate under two sets of conditions. The resulting oligomeric species have different degrees of cytotoxicity when added to the extracellular medium of the cells, as assessed by the extent of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, apoptosis, and influx of Ca2+ into the cells. The structural properties of the oligomeric variants were characterized by evaluating their surface hydrophobicity with 8-anilinonaphthalene-1-sulfonate (ANS) binding and by measuring their size by means of turbidimetry as well as light scattering. We find that increases in the surface hydrophobicity of the oligomers following mutation can promote the formation of larger assemblies and that the overall toxicity correlates with a combination of both surface hydrophobicity and size, with the most toxic oligomers having high hydrophobicity and small size. These results have allowed the relationships between these three parameters to be studied simultaneously and quantitatively, and have enabled the generation of an equation that is able to rationalize and even predict toxicity of the oligomers resulting from their surface hydrophobicity and size.

The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase.

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes.

Structure, activity, and inhibition of the Carboxyltransferase ß-subunit of acetyl coenzyme A carboxylase (AccD6) from Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, the carboxylation of acetyl coenzyme A (acetyl-CoA) to produce malonyl-CoA, a building block in long-chain fatty acid biosynthesis, is catalyzed by two enzymes working sequentially: a biotin carboxylase (AccA) and a carboxyltransferase (AccD). While the exact roles of the three different biotin carboxylases (AccA1 to -3) and the six carboxyltransferases (AccD1 to -6) in M. tuberculosis are still not clear, AccD6 in complex with AccA3 can synthesize malonyl-CoA from acetyl-CoA. A series of 10 herbicides that target plant acetyl-CoA carboxylases (ACC) were tested for inhibition of AccD6 and for whole-cell activity against M. tuberculosis. From the tested herbicides, haloxyfop, an arylophenoxypropionate, showed in vitro inhibition of M. tuberculosis AccD6, with a 50% inhibitory concentration (IC50) of 21.4 ± 1 µM. Here, we report the crystal structures of M. tuberculosis AccD6 in the apo form (3.0 Å) and in complex with haloxyfop-R (2.3 Å). The structure of M. tuberculosis AccD6 in complex with haloxyfop-R shows two molecules of the inhibitor bound on each AccD6 subunit. These results indicate the potential for developing novel therapeutics for tuberculosis based on herbicides with low human toxicity.

QM/MM study of the reaction mechanism of the carboxyl transferase domain of pyruvate carboxylase from Staphylococcus aureus.

Pyruvate carboxylase (PC) catalyzes the carboxylation of pyruvate to produce oxaloacetate. Its activity is directly related to insulin release and thus PC has recently attracted great interest as a potential target for diabetes treatment. In this article, the catalytic mechanism of the carboxyl transferase domain of PC from Staphylococcus aureus was investigated by using a combined quantum-mechanical/molecular-mechanical approach. Our calculation results indicate that the catalytic reaction starts from the decarboxylation of carboxybiotin to generate an enol-BTI intermediate, followed by two consecutive proton-transfer processes (from T908 to enol-BTI and from PYR to T908). During the catalytic reaction, the main-chain amide of T908 plays a key role in catching CO2 and preventing its diffusion from the active center. A triad of residues, R571, Q575, and K741, contributes both to substrate binding and enol-pyruvate stabilization. The oxyanion hole, consisting of the side-chain hydroxyl of S911 and the side-chain amino of Q870, plays an important role in stabilizing the hydroxyl anion of BTI. The coordination of the metal cation by pyruvate is a second sphere, rather than an inner sphere, interaction, and the metal cation stabilizes the species through the medium of residue K741. The decarboxylation of carboxybiotin corresponds to the highest free energy barrier of 21.7 kcal/mol. Our results may provide useful information for both the regulation of enzyme activity and the development of related biocatalytic applications.

Computational redesign of bacterial biotin carboxylase inhibitors using structure-based virtual screening of combinatorial libraries.

As the spread of antibiotic resistant bacteria steadily increases, there is an urgent need for new antibacterial agents. Because fatty acid synthesis is only used for membrane biogenesis in bacteria, the enzymes in this pathway are attractive targets for antibacterial agent development. Acetyl-CoA carboxylase catalyzes the committed and regulated step in fatty acid synthesis. In bacteria, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. Fragment-based screening revealed that amino-oxazole inhibits biotin carboxylase activity and also exhibits antibacterial activity against Gram-negative organisms. In this report, we redesigned previously identified lead inhibitors to expand the spectrum of bacteria sensitive to the amino-oxazole derivatives by including Gram-positive species. Using 9,411 small organic building blocks, we constructed a diverse combinatorial library of 1.2×108 amino-oxazole derivatives. A subset of 9×106 of these compounds were subjected to structure-based virtual screening against seven biotin carboxylase isoforms using similarity-based docking by eSimDock. Potentially broad-spectrum antibiotic candidates were selected based on the consensus ranking by several scoring functions including non-linear statistical models implemented in eSimDock and traditional molecular mechanics force fields. The analysis of binding poses of the top-ranked compounds docked to biotin carboxylase isoforms suggests that: (1) binding of the amino-oxazole anchor is stabilized by a network of hydrogen bonds to residues 201, 202 and 204; (2) halogenated aromatic moieties attached to the amino-oxazole scaffold enhance interactions with a hydrophobic pocket formed by residues 157, 169, 171 and 203; and (3) larger substituents reach deeper into the binding pocket to form additional hydrogen bonds with the side chains of residues 209 and 233. These structural insights into drug-biotin carboxylase interactions will be tested experimentally in in vitro and in vivo systems to increase the potency of amino-oxazole inhibitors towards both Gram-negative as well as Gram-positive species.

Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity.

Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N' of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.

Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs.

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates.

Effect of osmolytes on the fibrillation of HypF-N.

We have studied the effect of a series of stabilizing and destabilizing osmolytes on the fibrillation pattern of a model amyloidogenic protein, HypF-N. Under mildly denaturing conditions, HypF-N forms cross ß-sheet structures, characteristic of amyloid fibrils. In the presence of all stabilizing osmolytes except proline, fibrillation of HypF-N is inhibited. Notably, fibrillation kinetics is retarded at subdenaturing concentrations of chaotropes. In case of proline, fibrillation of HypF-N is accelerated. Thus, the changes during exposure of a protein to denaturing conditions in the presence of osmolyes cannot be extrapolated from their role as anti-fibrillation agents.

Correlation between Al(3+) -induced thermal stability and inhibition of fibrillation of N-terminal domain of the hydrogenase maturation factor.

Fibrillation can be induced in proteins by altering solvent conditions. Stabilization of the protofibrillar structure arrests formation of longer fibers. Thermal stability and fibrillation of N-terminal domain of the hydrogenase maturation factor (HypF-N) were studied in the presence of a series of metal ions. Only Al(3+) was able to reverse the thermal denaturation of HypF-N upon heating. On being exposed to denaturing conditions, the native protein formed fibrillar structure under moderately denaturing conditions, whereas in the presence of Al(3+) , the protein was found to retain its native conformation. Under strongly denaturing conditions, only Al(3+) was able to stabilize the protein in the fibrillar state. Spectrofluorimetric analysis revealed that Al(3+) alone was able to stabilize the partially unfolded intermediate state of HypF-N. Based on the similarity in observations, we propose a link between reversal of thermal instability of HypF-N and its ability to form an intermediate structure in the presence of Al(3+) . Al(3+) stabilizes the partially unfolded state in the N↔I↔U equilibrium so that upon heating, the three-dimensional structure of the protein is not lost completely. Kinetic analysis confirmed that Al(3+) interacts with an early structure on the aggregation landscape and delays fibrillation. Under mildly denaturing state, HypF-N is able to recover its native conformation in the presence of Al(3+) and under strongly denaturing conditions, the protein does not acquire a completely disordered structure. Instead, it forms an ordered ß-sheet-rich structure.