Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants.

The unfolding and refolding properties of human lysozyme and two amyloidogenic variants (Ile56Thr and Asp67His) have been studied by stopped-flow fluorescence and hydrogen exchange pulse labeling coupled with mass spectrometry. The unfolding of each protein in 5.4 M guanidine hydrochloride (GuHCl) is well described as a two-state process, but the rates of unfolding of the Ile56Thr variant and the Asp67His variant in 5.4 M GuHCl are ca. 30 and 160 times greater, respectively, than that of the wild type. The refolding of all three proteins in 0.54 M GuHCl at pH 5.0 proceeds through persistent intermediates, revealed by multistep kinetics in fluorescence experiments and by the detection of well-defined populations in quenched-flow hydrogen exchange experiments. These findings are consistent with a predominant mechanism for refolding of human lysozyme in which one of the structural domains (the alpha-domain) is formed in two distinct steps and is followed by the folding of the other domain (the beta-domain) prior to the assembly of the two domains to form the native structure. The refolding kinetics of the Asp67His variant are closely similar to those of the wild-type protein, consistent with the location of this mutation in an outer loop of the beta-domain which gains native structure only toward the end of the refolding process. By contrast, the Ile56Thr mutation is located at the base of the beta-domain and is involved in the domain interface. The refolding of the alpha-domain is unaffected by this substitution, but the latter has the effect of dramatically slowing the folding of the beta-domain and the final assembly of the native structure. These studies suggest that the amyloidogenic nature of the lysozyme variants arises from a decrease in the stability of the native fold relative to partially folded intermediates. The origin of this instability is different in the two variants, being caused in one case primarily by a reduction in the folding rate and in the other by an increase in the unfolding rate. In both cases this results in a low population of soluble partially folded species that can aggregate in a slow and controlled manner to form amyloid fibrils.     

Structural characterisation of the human alpha-lactalbumin molten globule at high temperature

Molten globules are partially folded forms of proteins thought to be general intermediates in protein folding. The 15N-1H HSQC NMR spectrum of the human alpha-lactalbumin (alpha-LA) molten globule at pH 2 and 20 degrees C is characterised by broad lines which make direct study by NMR methods difficult; this broadening arises from conformational fluctuations throughout the protein on a millisecond to microsecond timescale. Here, we find that an increase in temperature to 50 degrees C leads to a dramatic sharpening of peaks in the 15N-1H HSQC spectrum of human alpha-LA at pH 2. Far-UV CD and ANS fluorescence experiments demonstrate that under these conditions human alpha-LA maintains a high degree of helical secondary structure and the exposed hydrophobic surfaces that are characteristic of a molten globule. Analysis of the H(alpha), H(N) and 15N chemical shifts of the human alpha-LA molten globule at 50 degrees C leads to the identification of regions of native-like helix in the alpha-domain and of non-native helical propensity in the beta-domain. The latter may be responsible for the observed overshoot in ellipticity at 222 nm in kinetic refolding experiments.     

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.     

Rapid collapse and slow structural reorganisation during the refolding of bovine alpha-lactalbumin.

The refolding of bovine alpha-lactalbumin (BLA) from its chemically denatured state in 6 M GuHCl has been investigated by a variety of complementary biophysical approaches. CD experiments indicate that the species formed in the early stages of refolding of the apo-protein have at least 85 % of the alpha-helical content of the native state, and kinetic NMR experiments show that they possess near-native compactness. Hydrogen exchange measurements using mass spectrometry and NMR indicate that persistent structure in these transient species is located predominantly in the alpha-domain of the native protein and is similar to that present in the partially folded A-state formed by the protein at low pH. The extent of the exchange protection is, however, small, and there is no evidence for the existence of well-defined discrete kinetic intermediates of the type populated in the refolding of the structurally homologous c-type lysozymes. Rather, both mass spectrometric and NMR data indicate that the rate-determining transition from the compact partially structured (molten globule) species to the native state is highly cooperative. The data show that folding in the presence of Ca2+ is similar to that in its absence, although the rate is increased by more than two orders of magnitude. Sequential mixing experiments monitored by fluorescence spectroscopy indicate that this slower folding is not the result of the accumulation of kinetically trapped species. Rather, the data are consistent with a model in which binding of Ca2+ stabilizes native-like contacts in the partially folded species and reduces the barriers for the conversion of the protein to its native state. Taken together the results indicate that folding of BLA, in the presence of its four disulphide bonds, corresponds to one of the limiting cases of protein folding in which rapid collapse to a globule with a native-like fold is followed by a search for native-like side-chain contacts that enable efficient conversion to the close packed native structure.     

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.     

Intimate view of a kinetic protein folding intermediate: residue-resolved structure, interactions, stability, folding and unfolding rates, homogeneity

A cytochrome c kinetic folding intermediate was studied by hydrogen exchange (HX) pulse labeling. Advances in the technique and analysis made it possible to define the structured and unstructured regions, equilibrium stability, and kinetic opening and closing rates, all at an amino acid-resolved level. The entire N-terminal and C-terminal helices are formed and docked together at their normal native positions. They fray in both directions from the interaction region, due to a progression in both unfolding and refolding rates, leading to the surprising suggestion that helix propagation may proceed very slowly in the condensed milieu. Several native-like beta turns are formed. Some residues in the segment that will form the native 60s helix are protected but others are not, suggesting energy minimization to some locally non-native conformation in the transient intermediate. All other regions are unprotected, presumably dynamically disordered. The intermediate resembles a partially constructed native state. It is early, on-pathway, and all of the refolding molecules pass through it. These and related results consistently point to distinct, homogeneous, native-like intermediates in a stepwise sequential pathway, guided by the same factors that determine the native structure. Previous pulse labeling efforts have always assumed EX2 exchange during the labeling pulse, often leading to the suggestion of heterogeneous intermediates in alternative parallel pathways. The present work reveals a dominant role for EX1 exchange in the high pH labeling pulse, which will mimic heterogeneous behavior when EX2 exchange is assumed. The general problem of homogeneous versus heterogeneous intermediates and pathways is discussed.     

Native-like tertiary structure formation in the alpha-domain of a hen lysozyme two-disulfide variant.

Structure formation in two species of the two-disulfide variant of hen lysozyme was investigated by means of CD spectroscopy, disulfide exchange measurement, and 1H-NMR spectroscopy. One species, 2SS [6-127, 30-115], which contained the two disulfide bonds found in the alpha-domain of authentic lysozyme, had amounts of secondary and tertiary structures, and bacteriolytic activity comparable to those of authentic lysozyme, and showed a cooperative thermal unfolding. By contrast, the other species, 2SS [64-80, 76-94], which contained the beta-domain disulfide bond as well as the inter-domain one, had a limited amount of secondary structure and little tertiary structure. Disulfide-exchange did not occur for 2SS [6-127, 30-115], whereas it occurred for 2SS [64-80, 76-94], indicating that the protein main-chain fold coupled with the formation of two disulfide bonds is relatively stable for the former variant, while unstable for the latter. 1H-NMR spectra of 2SS [6-127, 30-115] showed that native-like local environment is present within the region that corresponds to the alpha-domain, while it is absent within the region that corresponds to the beta or inter-domain. These results indicate that the alpha-domain of hen lysozyme can be an independent folding domain at equilibrium. Although the bipartite nature in the structure formation of hen lysozyme is similar to that reported for alpha-lactalbumin, differences exist between the disulfide-intermediates of the two proteins in terms of the structural domain that accomplishes tertiary structure.     

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.     

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.     

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.     

Influence of Trp mutation on native, intermediate, and transition states of goat alpha-lactalbumin: an equilibrium and kinetic study

Equilibrium circular dichroism and kinetic stopped-flow fluorescence studies on the stability and the folding kinetics of a set of Trp to Phe mutants of goat alpha-lactalbumin (GLA) were used to characterize the native, intermediate, and transition states of these constructs. GLA contains four tryptophan residues, three of which, Trp26, Trp104, and Trp118, are located in the alpha-domain, while the fourth, Trp60, is located in the beta-domain. Trp26, Trp60, and Trp104 are part of a hydrophobic cluster, whereas Trp118 is situated in a more flexible region near the C-terminal end of the protein. In each case, the mutation leads to a reduction in the overall stability, but only for W26F and W60F is an equilibrium intermediate observed in guanidine hydrochloride-induced unfolding experiments. In kinetic refolding experiments, however, for all samples a burst phase is observed, the amplitude of which depends on the specific mutation. Refolding and unfolding kinetics can adequately be described by a sequential three-state mechanism. phi value analysis showed that the local structure around Trp26, Trp60, and Trp104 is formed in the intermediate and in the transition state of the folding reaction, while around Trp118 no persistent native contacts are observed. From these findings, we conclude that, although hydrophobicity is a major driving force for folding, minor steric changes induced by point mutation can considerably influence the overall stability and the folding process of the protein.     

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.     

Evidence concerning rate-limiting steps in protein folding from the effects of trifluoroethanol

The refolding kinetics of 13 proteins have been studied in the presence of 2,2,2-trifluoroethanol (TFE). Low concentrations of TFE increased the folding rates of all the proteins, whereas higher concentrations have the opposite effect. The extent of deceleration of folding correlates closely with similar effects of guanidine hydrochloride and can be related to the burial of accessible surface area during folding. For those proteins folding in a two-state manner, the extent of acceleration of folding correlates closely with the number of local backbone hydrogen bonds in the native structure. For those proteins that fold in a multistate manner, however, the extent of acceleration is much smaller than that predicted from the data for two-state proteins. These results support the concept that for two-state proteins the search for native-like contacts is a key aspect of the folding reaction, whereas the rate-determining steps for folding of multistate proteins are associated with the reorganization of stable structure within a collapsed state or with the search for native-like interactions within less structured regions.     

Specific non-native hydrophobic interactions in a hidden folding intermediate: implications for protein folding

Structures of intermediates and transition states in protein folding are usually characterized by amide hydrogen exchange and protein engineering methods and interpreted on the basis of the assumption that they have native-like conformations. We were able to stabilize and determine the high-resolution structure of a partially unfolded intermediate that exists after the rate-limiting step of a four-helix bundle protein, Rd-apocyt b(562), by multidimensional NMR methods. The intermediate has partial native-like secondary structure and backbone topology, consistent with our earlier native state hydrogen exchange results. However, non-native hydrophobic interactions exist throughout the structure. These and other results in the literature suggest that non-native hydrophobic interactions may occur generally in partially folded states. This can alter the interpretation of mutational protein engineering results in terms of native-like side chain interactions. In addition, since the intermediate exists after the rate-limiting step and Rd-apocyt b(562) folds very rapidly (k(f) approximately 10(4) s(-1)), these results suggest that non-native hydrophobic interactions, in the absence of topological misfolding, are repaired too rapidly to slow folding and cause the accumulation of folding intermediates. More generally, these results illustrate an approach for determining the high-resolution structure of folding intermediates.     

The role of Trp-82 in the folding of intestinal fatty acid binding protein

Multiple phases have been observed during the folding and unfolding of intestinal fatty acid binding protein (WT-IFABP) by stopped-flow fluorescence. Site-directed mutagenesis has been used to examine the role of each of the two tryptophans of this protein in these processes. The unfolding and refolding kinetics of the mutant protein containing only tryptophan 82 (W6Y-IFABP) showed that the tryptophan at this location was critical to the fluorescence signal changes observed throughout the unfolding reaction and early in the refolding reaction. However, the kinetic patterns of the mutant protein containing only tryptophan 6 (W82Y-IFABP) indicated that the tryptophan at this location participated in the fluorescence signal changes observed early in the unfolding reaction and late in the refolding reaction. Together, these data suggest that native-like structure was formed first in the vicinity of tryptophan 82, near the center of the hydrophobic core of this beta-sheet protein, prior to formation of native-like structure in the periphery of the protein.     

Microsecond folding of the cold shock protein measured by a pressure-jump technique

A pressure-jump apparatus was employed in investigating the kinetics of protein unfolding and refolding. In the reaction cell, the pressure can be increased or decreased by 100-160 bar within 50-100 microseconds and then held constant. Thus, unfolding and refolding reactions in the time range from 70 microseconds to 70 s can be followed with this technique. Measurements are possible in the transition regions of thermally or denaturant-induced folding in a wide range of temperatures and solvent conditions. We used this pressure-jump method to determine the temperature dependence of the rate constants of unfolding and refolding of the cold shock protein of Bacillus subtilis and of three variants thereof with Phe --> Ala substitutions in the central beta-sheet region. For all variants, the change in heat capacity occurred in refolding between the unfolded and activated states, suggesting that the overall native-like character of the activated state of folding was not changed by the deletion of individual Phe side chains. The Phe27Ala mutation affected the rate of unfolding only; the Phe15Ala and Phe17Ala mutations changed the kinetics of both unfolding and refolding. Although the activated state of folding of the cold shock protein is overall native-like, individual side chains are still in a non-native environment.     

Characterization of the unfolded state of bovine alpha-lactalbumin and comparison with unfolded states of homologous proteins

The unfolded states of three homologous proteins with a very similar fold have been investigated by heteronuclear NMR spectroscopy. Secondary structure propensities as derived from interpretation of chemical shifts and motional restrictions as evidenced by heteronuclear (15)N relaxation rates have been analyzed in the reduced unfolded states of hen lysozyme and the calcium-binding proteins bovine alpha-lactalbumin and human alpha-lactalbumin. For all three proteins, significant deviations from random-coil predictions can be identified; in addition, the unfolded states also differ from each other, despite the fact that they possess very similar structures in their native states. Deviations from random-coil motional properties are observed in the alpha- and the beta-domain in bovine alpha-lactalbumin and lysozyme, while only regions within the alpha-domain deviate in human alpha-lactalbumin. The motional restrictions and residual secondary structure are determined both by the amino acid sequence of the protein and by residual long-range interactions. Even a conservative single point mutation from I to L in a highly conserved region between the two alpha-lactalbumins results in considerable differences in the motional properties. Given the differences in oxidative folding between hen lysozyme and alpha-lactalbumin, the results obtained on the unfolded states suggest that residual long-range interactions, i.e., those between the alpha- and the beta-domain of lysozyme, may act as nucleation sites for protein folding, while this property of residual structure is replaced by the calcium-binding site between the domains in alpha-lactalbumin.     

Hydrophobic photolabeling as a new method for structural characterization of molten globule and related protein folding intermediates.

Recent advances in attempts to unravel the protein folding mechanism have indicated the need to identify the folding intermediates. Despite their transient nature, in a number of cases it has been possible to detect and characterize some of the equilibrium intermediates, for example, the molten globule (MG) state. The key features of the MG state are retention of substantial secondary structure of the native state, considerable loss of tertiary structure leading to increased hydrophobic exposure, and a compact structure. NMR, circular dichroism, and fluorescence spectroscopies have been most useful in characterizing such intermediates. We report here a new method for structural characterization of the MG state that involves probing the exposed hydrophobic sites with a hydrophobic photoactivable reagent--2[3H]diazofluorene. This carbene-based reagent binds to hydrophobic sites, and on photolysis covalently attaches itself to the neighboring amino acid side chains. The reagent photolabels alpha-lactalbumin as a function of pH (3-7.4), the labeling at neutral pH being negligible and maximal at pH 3. Chemical and proteolytic fragmentation of the photolabeled protein followed by peptide sequencing permitted identification of the labeled residues. The results obtained indicate that the sequence corresponding to B (23-34) and C (86-98) helix of the native structure are extensively labeled. The small beta-domain (40-50) is poorly labeled, Val42 being the only residue that is significantly labeled. Our data, like NMR data, indicate that in the MG state of alpha-lactalbumin, the alpha-domain has a greater degree of persistent structure than the beta-domain. However, unlike the NMR method, the photolabeling method is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in folding intermediates as the reagent binds and on photolysis covalently links to these regions.     

Intestinal fatty acid binding protein: the folding mechanism as determined by NMR studies

The intestinal fatty acid binding protein is composed of two beta-sheets surrounding a large interior cavity. There is a small helical domain associated with the portal for entry of the ligand into the cavity. Denaturation of the protein has been monitored in a residue-specific manner by collecting a series of two-dimensional (1)H-(15)N heteronuclear single-quantum coherence (HSQC) NMR spectra from 0 to 6.5 M urea under equilibrium conditions. In addition, rates for hydrogen-deuterium exchange have been measured as a function of denaturant concentration. Residual, native-like structure persists around hydrophobic clusters at very high urea concentrations. This residual structure (reflecting only about 2-7% persistence of native-like structure) involves the turns between beta-strands and between the two short helices. If this persistence is assumed to reflect transient native-like structure in these regions of the polypeptide chain, these sites may serve as nucleation sites for folding. The data obtained at different urea concentrations are then analyzed on the basis of peak intensities relative to the intensities in the absence of urea reflecting the extent of secondary structure formation. At urea concentrations somewhat below 6.5 M, specific hydrophobic residues in the C-terminal beta-sheet interact and two strands, the D and E strands in the N-terminal beta-sheet, are stabilized. These latter strands surround one of the turns showing residual structure. With decreasing urea concentrations, the remaining strands are stabilized in a specific order. The early strand stabilization appears to trigger the formation of the remainder of the C-terminal beta-sheet. At low urea concentrations, hydrogen bonds are formed. A pathway is proposed on the basis of the data describing the early, intermediate, and late folding steps for this almost all beta-sheet protein. The data also show that there are regions of the protein which appear to act in a concerted manner at intermediate steps in refolding.     

Crystal structure of a novel bacterial cell-surface flagellin binding to a polysaccharide

Bacterial flagellins are generally self-assembled into extracellular flagella for cell motility. However, the flagellin homologue p5 is found on the cell surface of Sphingomonas sp. A1 (strain A1) and binds tightly to the alginate polysaccharide. To assimilate alginate, strain A1 forms a mouthlike pit on the cell surface and concentrates the polymer in the pit. p5 is a candidate receptor that recognizes extracellular alginate and controls pit formation. To improve our understanding of the structure and function of p5, we determined the crystal structure of truncated p5 (p5DeltaN53C45) at 2.0 A resolution. This, to our knowledge, is the first structure of flagellin_IN motif-containing flagellin. p5DeltaN53C45 consists of two domains: an alpha-domain rich in alpha-helices that forms the N- and C-terminal regions and a beta-domain rich in beta-strands that constitutes the central region. The alpha-domain is structurally similar to the D1 domain of Salmonella typhimurium flagellin, while the beta-domain is structurally similar to the finger domain of the bacteriophage T4 baseplate protein that is important for intermolecular interactions between baseplate and a long or short tail fiber. Results from the deletion mutant analysis suggest that residues 20-40 and 353-363 are responsible for alginate binding. Truncated N- and C-terminal regions are thought to constitute alpha-helices extending from the alpha-domain. On the basis of the size and surface charge, the cleft in extended alpha-helices is proposed as an alginate binding site of p5. Structural similarity in the beta-domain suggests that the beta-domain is involved in the proper localization and/or orientation of p5 on the cell surface.