Unfolding of hen egg lysozyme by molecular dynamics simulations at 300K: insight into the role of the interdomain interface

We present the results of two 1.2 ns molecular dynamics (MD) unfolding simulations on hen egg lysozyme in water at 300K, performed using a new procedure called PEDC (Path Exploration With Distance Constraints). This procedure allows exploration of low energy structures as a function of increasing RMSD from the native structure, and offers especially the possibility of extensive exploration of the conformational space during the initial unfolding stages. The two independent MD simulations gave similar chronology of unfolding events: disruption of the active site, kinking of helix C, partial unfolding of the three-stranded beta-sheet to a two-stranded sheet (during which the helices A, B, and D remain to a great extent native), and finally unfolding of the beta-domain and partial unfolding of the alpha-domain in which hydrophobic clusters persist. We show particularly that the loss of hydrophobic contacts between the beta-sheet turn residues Leu55 and Ile56 and the hydrobic patch of the alpha-domain destabilizes the beta-domain and leads to its unfolding, suggesting that the correct embedding of these residues in the alpha-beta interface may constitute the rate limiting step in folding. These results are in accord with experimental observations on the folding/unfolding behavior of hen egg lysozyme at room temperature. They would also explain the loss of stability and the tendency to aggregation observed for the mutant Leu55Thr, and the slow refolding kinetics observed in the analogous amyloidogenic variant of human lysozyme.     

Oxidative refolding of amyloidogenic variants of human lysozyme

The oxidative refolding of human lysozyme and its two best characterised amyloidogenic variants, Ile56Thr and Asp67His, has been investigated in vitro by means of the concerted application of a range of biophysical techniques. The results show that in each case the ensemble of reduced denatured conformers initially collapses into a large number of unstructured intermediates with one or two disulphide bonds, the majority of which then fold to form the native-like three-disulphide intermediate, des-[77-95]. The slow step in the overall folding reaction involves the rearrangement of the latter to the fully oxidised native protein containing four disulphide bonds. The Ile56Thr and Asp67His variants were found to fold faster than the wild-type protein by a factor of 2 and 3 respectively, an observation that can be attributed primarily to the reduction in the barriers to conformational rearrangements that results from both the mutations. The efficient folding of these variants despite their enhanced propensities to aggregate when compared to the wild-type protein is consistent with their ability to be secreted in sufficient quantities to give rise to the systemic amyloidoses with which they are associated.     

Insights into the conformational changes of several human lysozyme variants associated with hereditary systemic amyloidosis

In this study, various ethanol- and temperature-induced molecular dynamics simulations were conducted to investigate the conformational changes of several human lysozyme variants (I56T, D67H, and T70N) associated with hereditary systemic amyloidosis. The results show that these variants are all more sensitive to conditions affecting the structural integrity of this protein. The structural analyses of these variants reveal a high population of more unstable beta-domain and distorted hydrophobic core compared to the wild-type human lysozyme, particularly for the two natural amyloidogenic variants D67H and I56T. For the D67H variant, the distance between the mass centers of residues 54 and 67 was found to elongate as a result of the destruction of the hydrogen-bonding network stabilizing the two long loops in the beta-domain. It further accelerates the unfolding of this variant, starting from the hydrophobic core between the alpha- and beta-domains. For the I56T variant, the introduction of a hydrophilic residue in the hydrophobic core directly destroys the native contacts in the alpha-beta interface, leading to fast unfolding. The present results are consistent with the previous hypothesis suggesting that the distortion of the hydrophobic core at the alpha-beta interface putatively results in the formation of the initial "seed" for amyloid fibrils.     

Rationalising lysozyme amyloidosis: insights from the structure and solution dynamics of T70N lysozyme

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 6.5A. 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 beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-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.     

Amyloid fibril formation and seeding by wild-type human lysozyme and its disease-related mutational variants

Wild-type human lysozyme and its two stable amyloidogenic variants have been found to form partially folded states at low pH. These states are characterized by extensive disruption of tertiary interactions and partial loss of secondary structure. Incubation of the proteins at pH 2.0 and 37 degrees C (Ile56Thr and Asp67His variants) or 57 degrees C (wild-type) results in the formation of large numbers of fibrils over several days of incubation. Smaller numbers of fibrils could be observed under other conditions, including neutral pH. These fibrils were analyzed by electron microscopy, Congo red birefringence, thioflavine-T binding, and X-ray fiber diffraction, which unequivocally show their amyloid character. These data demonstrate that amyloidogenicity is an intrinsic property of human lysozyme and does not require the presence of specific mutations in its primary structure. The amyloid fibril formation is greatly facilitated, however, by the introduction of "seeds" of preformed fibrils to the solutions of the variant proteins, suggesting that seeding effects could be important in the development of systemic amyloidosis. Fibril formation by wild-type human lysozyme is greatly accelerated by fibrils of the variant proteins and vice versa, showing that seeding is not specific to a given protein. The fact that wild-type lysozyme has not been found in ex vivo deposits from patients suffering from this disease is likely to be related to the much lower population of incompletely folded states for the wild-type protein compared to its amyloidogenic variants under physiological conditions. These results support the concept that the ability to form amyloid is a generic property of proteins, but one that is mitigated against in a normally functioning organism.     

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.     

Local cooperativity in the unfolding of an amyloidogenic variant of human lysozyme

Hydrogen exchange experiments monitored by NMR and mass spectrometry reveal that the amyloidogenic D67H mutation in human lysozyme significantly reduces the stability of the beta-domain and the adjacent C-helix in the native structure. In addition, mass spectrometric data reveal that transient unfolding of these regions occurs with a high degree of cooperativity. This behavior results in the occasional population of a partially structured intermediate in which the three alpha-helices that form the core of the alpha-domain still have native-like structure, whereas the beta-domain and C-helix are simultaneously substantially unfolded. This finding suggests that the extensive intermolecular interactions that will be possible in such a species are likely to initiate the aggregation events that ultimately lead to the formation of the well-defined fibrillar structures observed in the tissues of patients carrying this mutation in the lysozyme gene.     

Kinetic evidence of an on-pathway intermediate in the folding of lysozyme

By means of a kinetic test, it was demonstrated that one of the folding intermediates (Ialpha) of hen lysozyme with alpha-domain folded and beta-domain unfolded is on the folding pathway under the classical definition. Ialpha folds to the native (N) state directly (unfolded (U) <==> Ialpha <==> N) without having to unfold to U and then refold to N through alternative folding pathways as in Ialpha <==> U <==> N.     

Thermal unfolding of an intermediate is associated with non-Arrhenius kinetics in the folding of hen lysozyme

A variety of techniques, including quenched-flow hydrogen exchange labelling monitored by electrospray ionization mass spectrometry, and stopped-flow absorbance, fluorescence and circular dichroism spectroscopy, has been used to investigate the refolding kinetics of hen lysozyme over a temperature range from 2 degrees C to 50 degrees C. Simple Arrhenius behaviour is not observed, and although the overall rate of folding increases from 2 to 40 degrees C, it decreases above 40 degrees C. In addition, the transient intermediate on the major folding pathway at 20 degrees C, in which the alpha-domain is persistently structured in the absence of a stable beta-domain, is thermally unfolded in a sigmoidal transition (T(m) approximately 40 degrees C) indicative of a cooperatively folded state. At all temperatures, however, there is evidence for fast ( approximately 25 %) and slow ( approximately 75 %) populations of refolding molecules. By using transition state theory, the kinetic data from various experiments were jointly fitted to a sequential three-state model for the slow folding pathway. Together with previous findings, these results indicate that the alpha-domain intermediate is a productive species on the folding route between the denatured and native states, and which accumulates as a consequence of its intrinsic stability. Our analysis suggests that the temperature dependence of the rate constant for lysozyme folding depends on both the total change in the heat capacity between the ground and transition states (the dominant factor at low temperatures) and the heat-induced destabilization of the alpha-domain intermediate (the dominant factor at high temperatures). Destabilization of such kinetically competent intermediate species is likely to be a determining factor in the non-Arrhenius temperature dependence of the folding rate of those proteins for which one or more intermediates are populated.     

In vivo glycosylation suppresses the aggregation of amyloidogenic hen egg white lysozymes expressed in yeast

The mutant hen egg white lysozymes Ile55Thr and Asp66His, corresponding to human amyloidogenic mutant lysozymes Ile56Thr and Asp67His, respectively, were secreted in Saccharomyces cerevisiae. The amyloidogenic mutants (I55T and D66H) of hen egg white lysozymes were remarkably less soluble than that of the wild-type protein. To enhance the secretion of these mutants, we constructed the glycosylated amyloidogenic lysozymes (I55T/G49N and D66H/G49N) having the N-glycosylation signal sequence (Asn-X-Ser) by the substitution of glycine with asparagine at position 49. The secretion of these glycosylated mutant proteins is greatly increased in S. cerevisiae, compared with that of non-glycosylated type. Both the glycosylated mutants retained about 40% enzymatic activity when incubated at pH 7.4 for 1 h at the physiological temperature of 37 degrees C whereas the non-glycosylated proteins eventually lost all activity under these conditions. These results suggest that the glycosylated chains could mask the beta-strand of amyloidogenic lysozymes from the intermolecular cross-beta-sheet association, thus improving the solubility of amyloidogenic lysozymes.     

Structural and folding dynamic properties of the T70N variant of human lysozyme

Definition of the transition mechanism from the native globular protein into fibrillar polymer was greatly improved by the biochemical and biophysical studies carried out on the two amyloidogenic variants of human lysozyme, I56T and D67H. Here we report thermodynamic and kinetic data on folding as well as structural features of a naturally occurring variant of human lysozyme, T70N, which is present in the British population at an allele frequency of 5% and, according to clinical and histopathological data, is not amyloidogenic. This variant is less stable than the wild-type protein by 3.7 kcal/mol, but more stable than the pathological, amyloidogenic variants. Unfolding kinetics in guanidine are six times faster than in the wild-type, but three and twenty times slower than in the amyloidogenic variants. Enzyme catalytic parameters, such as maximal velocity and affinity, are reduced in comparison to the wild-type. The solution structure, determined by 1H NMR and modeling calculations, exhibits a more compact arrangement at the interface between the beta-sheet domain and the subsequent loop on one side and part of the alpha domain on the other side, compared with the wild-type protein. This is the opposite of the conformational variation shown by the amyloidogenic variant D67H, but it accounts for the reduced stability and catalytic performance of T70N.     

Immunochemical pulsed-labeling characterization of intermediates during hen lysozyme oxidative folding

Previous studies have shown that reduced hen egg white lysozyme refolds and oxidizes according to a linear model, in which the number of disulfide bonds increases sequentially. In this study, we describe the kinetics of native tertiary structure formation during the oxidative-renaturation of reduced hen egg white lysozyme, as monitored using an immunochemical pulsed-labeling method based on enzyme-linked immunosorbent assay (ELISA) in conjunction with two monoclonal antibodies (mAb). Each of these antibodies recognizes a separate face of the native lysozyme surface and, more importantly, each epitope is composed of discontinuous regions of the polypeptide chain. Renaturation kinetics were studied under the same refolding conditions as previous investigations of the kinetics of the regain of far-UV CD, fluorescence, enzymatic activity, and disulfide bonds. Comparison of our results with the results from those studies showed that the immunoreactivity (i.e., the native fold) of the alpha-domain appeared in intermediates containing two SS bonds only (C6-C127 and C30-C115), while the immunoreactivity of the beta-domain appeared together with the formation of the third SS bond (C64-C80). Thus, the alpha-domain folds before the beta-domain during the oxidative folding of reduced lysozyme.     

NMR structural study of two-disulfide variant of hen lysozyme: 2SS[6-127, 30-115]--a disulfide intermediate with a partly unfolded structure

The 15N-labeled recombinant hen lysozyme and two species of two-disulfide variants, denoted as 2SS[6-127, 30-115] and 2SS[64-80, 76-94], were studied by means of NMR spectroscopy. The former variant contains two disulfide bridges in the alpha-domain, while the latter has one disulfide bridge in the beta-domain and the other one at the interface between two domains. Resonance assignments were performed using 3D TOCSY-HSQC and NOESY-HSQC spectra. The 15N-1H-HSQC spectrum of 2SS[6-127, 30-115] was similar to that of recombinant lysozyme as a whole, although a number of cross-peaks disappeared. On the other hand, the HSQC spectrum of 2SS[64-80, 76-94] was characteristic of unfolded proteins. The structure of 2SS[6-127, 30-115] was thoroughly examined on the basis of NOE contacts determined by NMR spectroscopy. The structure of the alpha-domain was quite similar to that of authentic lysozyme, while the beta-domain was largely unstructured. However, NMR data clearly demonstrated that some residual structures exist in the beta-domain. The beta1 and beta2 strands were maintained stably as an antiparallel beta-sheet. In addition, the residues 55 and 56 were located in the vicinity of the end of the B-helix. Further, the C-helix was properly set with side-chains of I88, V92, K96, and V99 facing toward the hydrophobic core in the alpha-domain. These residual structures inherent in the amino acid sequence were evaluated concerning the folding process of lysozyme. Our experiments imply that the establishment of the backbone conformation ranging from residues 76-99 plays a key role in attaining the cooperativity between two domains required for the folding transition.     

Characterization of the structure and dynamics of amyloidogenic variants of human lysozyme by NMR spectroscopy

The structures and dynamics of the native states of two mutational variants of human lysozyme, I56T and D67H, both associated with non-neuropathic systemic amyloidosis, have been investigated by NMR spectroscopy. The (1)H and (15)N main-chain amide chemical shifts of the I56T variant are very similar to those of the wild-type protein, but those of the D67H variant are greatly altered for 28 residues in the beta-domain. This finding is consistent with the X-ray crystallographic analysis, which shows that the structure of this variant is significantly altered from that of the wild-type protein in this region. The (1)H-(15)N heteronuclear NOE values show that, with the exception of V121, every residue in the wild-type and I56T proteins is located in tightly packed structures characteristic of the native states of most proteins. In contrast, D67H has a region of substantially increased mobility as shown by a dramatic decrease in heteronuclear NOE values of residues near the site of mutation. Despite this unusual flexibility, the D67H variant has no greater propensity to form amyloid fibrils in vivo or in vitro than has I56T. This finding indicates that it is the increased ability of the variants to access partially folded conformations, rather than intrinsic changes in their native state properties, that is the origin of their amyloidogenicity.     

Localized nature of the transition-state structure in goat alpha-lactalbumin folding

To investigate whether the structure partially formed in the molten globule folding intermediate of goat alpha-lactalbumin is further organized in the transition state of folding, we constructed a number of mutant proteins and performed Phi-value analysis on them. For this purpose, we measured the equilibrium unfolding transitions and kinetic refolding and unfolding reactions of the mutants using equilibrium and stopped-flow kinetic circular dichroism techniques. The results show that the mutants with mutations located in the A-helix (V8A, L12A), the B-helix (V27A), the beta-domain (L52A, W60A), the C-helix (K93A, L96A), the C-D loop (Y103F), the D-helix (L105A, L110A), and the C-terminal 3(10)-helix (W118F), have low Phi-values, less than 0.2. On the other hand, D87N, which is located on the Ca(2+)-binding site, has a high Phi-value, 0.91, indicating that tight packing of the side-chain around Asp87 occurs in the transition state. One beta-domain mutant (I55V) and three C-helix mutants (I89V, V90A, and I95V) demonstrated intermediate Phi-values, between 0.4 and 0.7. These results indicate that the folding nucleus in the transition state of goat alpha-LA is not extensively distributed over the alpha-domain of the protein, but very localized in a region that contains the Ca(2+)-binding site and the interface between the C-helix and the beta-domain. This is apparently in contrast with the fact that the molten globule state of alpha-lactalbumin has a partially formed structure inside the alpha-domain. It is concluded that the specific docking of the alpha and beta-domains at a domain interface is necessary for this protein to organize its native structure from the molten globule intermediate.     

Reversible denaturation of the gene V protein of bacteriophage f1

The guanidine hydrochloride (GuHCl)-induced denaturation of the gene V protein of bacteriophage f1 has been studied, using the chemical reactivity of a cysteine residue that is buried in the folded protein and the circular dichroism (CD) at 211 and 229 nm as measures of the fraction of polypeptide chains in the folded form. It is found that this dimeric protein unfolds in a single cooperative transition from a folded dimer to two unfolded monomers. A folded, monomeric form of the gene V protein was not detected at equilibrium. The kinetics of unfolding of the gene V protein in 3 M GuHCl and the refolding in 2 M GuHCl are also consistent with a transition between a folded dimer and two unfolded monomers. The GuHCl concentration dependence of the rates of folding and unfolding suggests that the transition state for folding is near the folded conformation.     

Contributions of hydrophobic domain interface interactions to the folding and stability of human gammaD-crystallin

Human gammaD-crystallin (HgammaD-Crys) is a monomeric eye lens protein composed of two highly homologous beta-sheet domains. The domains interact through interdomain side chain contacts forming two structurally distinct regions, a central hydrophobic cluster and peripheral residues. The hydrophobic cluster contains Met43, Phe56, and Ile81 from the N-terminal domain (N-td) and Val132, Leu145, and Val170 from the C-terminal domain (C-td). Equilibrium unfolding/refolding of wild-type HgammaD-Crys in guanidine hydrochloride (GuHCl) was best fit to a three-state model with transition midpoints of 2.2 and 2.8 M GuHCl. The two transitions likely corresponded to sequential unfolding/refolding of the N-td and the C-td. Previous kinetic experiments revealed that the C-td refolds more rapidly than the N-td. We constructed alanine substitutions of the hydrophobic interface residues to analyze their roles in folding and stability. After purification from E. coli, all mutant proteins adopted a native-like structure similar to wild type. The mutants F56A, I81A, V132A, and L145A had a destabilized N-td, causing greater population of the single folded domain intermediate. Compared to wild type, these mutants also had reduced rates for productive refolding of the N-td but not the C-td. These data suggest a refolding pathway where the domain interface residues of the refolded C-td act as a nucleating center for refolding of the N-td. Specificity of domain interface interactions is likely important for preventing incorrect associations in the high protein concentrations of the lens nucleus.     

Reduced global cooperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations

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 alpha-helices that form the core of the alpha-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, and the 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.     

The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR^?, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.     

Independent nucleation and heterogeneous assembly of structure during folding of equine lysozyme.

The refolding of equine lysozyme from guanidinium chloride has been studied using hydrogen exchange pulse labelling in conjunction with NMR spectroscopy and stopped flow optical methods. The stopped flow optical experiments indicate that extensive hydrophobic collapse occurs rapidly after the initiation of refolding. Pulse labelling experiments monitoring nearly 50 sites within the protein have enabled the subsequent formation of native-like structure to be followed in considerable detail. They reveal that an intermediate having persistent structure within three of the four helices of the alpha-domain of the protein is formed for the whole population of molecules within 4 ms. Subsequent to this event, however, the hydrogen exchange protection kinetics are complex and highly heterogeneous. Analysis of the results by fitting to stretched exponential functions shows that a series of other intermediates is formed as a consequence of the stepwise assembly of independently nucleated local regions of structure. In some molecules the next step in folding involves the stabilisation of the remaining helix in the alpha-domain, whilst in others persistent structure begins to form in the beta-domain. The formation of native-like structure throughout the beta-domain is itself heterogeneous, involving at least three kinetically distinguishable steps. Residues in loop regions throughout the protein attain persistent structure more slowly than regions of secondary structure. There is in addition evidence for locally misfolded regions of structure that reorganise on much longer timescales. The results reveal that the native state of the protein is generated by the heterogeneous assembly of a series of locally cooperative regions of structure. This observation has many features in common with the findings of recent theoretical simulations of protein folding.