Role of individual disulfide bonds in hen lysozyme early folding steps

To probe the role of individual disulfide bonds in the folding kinetics of hen lysozyme, the variants with two mutations, C30A,C115A, C64A,C80A, and C76A,C94A, were constructed. The corresponding proteins, each lacking one disulfide bond, were produced in Escherichia coli as inclusion bodies and solubilized, purified, and renatured/oxidized using original protocols. Their enzymatic, spectral, and hydrodynamic characteristics confirmed that their conformations were very similar to that of native wild-type (WT) lysozyme. Stopped-flow studies on the renaturation of these guanidine-unfolded proteins with their three disulfides intact showed that, for the three variants, the native far-UV ellipticity was regained in a burst phase within the 4-ms instrument dead-time. The transient overshoots of far-UV ellipticity and tryptophan fluorescence that follow the burst phase, as well as the kinetics of transient 8-anilino-1-naphthalene-sulfonic acid (ANS) binding, were diversely affected depending on the variant. Together with previous reports on the folding kinetics of WT lysozyme carboxymethylated on cysteines 6 and 127, detailed analysis of the kinetics showed that (1) none of the disulfide bonds were indispensable for the rapid formation (<4 ms) of the native-like secondary structure; (2) the two intra-alpha-domain disulfides (C6-C127 and C30-C115) must be simultaneously present to generate the trapped intermediate responsible for the slow folding population observed in WT lysozyme; and (3) the intra-beta-domain (C64-C80) and the inter-alphabeta-domains (C76-C94) disulfides do not affect the kinetics of formation of the trapped intermediate but are involved in its stability.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme.

To assess the respective roles of local and long-range interactions during protein folding, the influence of the native disulfide bonds on the early formation of secondary structure was investigated using continuous-flow circular dichroism. Within the first 4 ms of folding, lysozyme with intact disulfide bonds already had a far-UV CD spectrum reflecting large amounts of secondary structure. Conversely, reduced lysozyme remained essentially unfolded at this early folding time. Thus, native disulfide bonds not only stabilize the cfinal conformation of lysozyme but also provide, in early folding intermediates, the necessary stabilization that favors the formation of secondary structure.     

Effect of ethanol on folding of hen egg-white lysozyme under acidic condition

The equilibrium and kinetics of folding of hen egg-white lysozyme were studied by means of CD spectroscopy in the presence of varying concentrations of ethanol under acidic condition. The equilibrium transition curves of guanidine hydrochloride-induced unfolding in 13 and 26% (v/v) ethanol have shown that the unfolding significantly deviates from a two-state mechanism. The kinetics of denaturant-induced refolding and unfolding of hen egg-white lysozyme were investigated by stopped-flow CD at three ethanol concentrations: 0, 13, and 26% (v/v). Immediately after dilution of the denaturant, the refolding curves showed a biphasic time course in the far-UV region, with a burst phase with a significant secondary structure and a slower observable phase. However, when monitored by the near-UV CD, the burst phase was not observed and all refolding kinetics were monophasic. To clarify the effect of nonnative secondary structure induced by the addition of ethanol on the folding/unfolding kinetics, the kinetic m values were estimated from the chevron plots obtained for the three ethanol concentrations. The data indicated that the folding/unfolding kinetics of hen lysozyme in the presence of varying concentrations of ethanol under acidic condition is explained by a model with both on-pathway and off-pathway intermediates of protein folding.     

Equilibrium and kinetic studies on folding of canine milk lysozyme

Thermal and chemical unfolding studies of the calcium-binding canine lysozyme (CL) by fluorescence and circular dichroism spectroscopy show that, upon unfolding in the absence of calcium ions, a very stable equilibrium intermediate state is formed. At room temperature and pH 7.5, for example, a stable molten globule state is attained in 3 M GdnHCl. The existence of such a pure and stable intermediate state allowed us to extend classical stopped-flow fluorescence measurements that describe the transition from the native to the unfolded form, with kinetic experiments that monitor separately the transition from the unfolded to the intermediate state and from the intermediate to the native state, respectively. The overall refolding kinetics of apo-canine lysozyme are characterized by a significant drop in the fluorescence intensity during the dead time, followed by a monoexponential increase of the fluorescence with k = 3.6 s(-1). Furthermore, the results show that, unlike its drastic effect on the stability, Ca(2+)-binding only marginally affects the refolding kinetics. During the refolding process of apo-CL non-native interactions, comparable to those observed in hen egg white lysozyme, are revealed by a substantial quenching of tryptophan fluorescence. The dissection of the refolding process in two distinct steps shows that these non-native interactions only occur in the final stage of the refolding process in which the two domains match to form the native conformation.     

Effect of the structure of the denatured state of lysozyme on the aggregation reaction at the early stages of folding from the reduced form

We previously demonstrated that the hydrophobic clusters present in hen lysozyme under denaturing conditions were disrupted by the mutation of Trp62 to Gly (W62G). In order to examine the effects of the structure of the denatured state of W62G lysozyme on folding, we analyzed the early events in the folding of reduced W62G lysozyme in detail. From the exchange measurements of disulfide bonds using the variants containing a pair of cysteine residues (1SS), it was found that the formation of disulfide bond in the W62G1SS lysozyme was not accompanied by a prominent interaction between amino acid residues, indicating that the disruption of the hydrophobic core led to the random folding at the early stages in the process of folding of the reduced lysozyme. On the other hand, analyses of the oxidative-renaturation of reduced W62G lysozymes, as well as measurements of the extent of aggregation of the reduced and carboxy amido methylated W62G lysozyme, indicated that the formation of an aggregate is more prominent in the reduced W62G lysozyme than in the reduced wild-type lysozyme. Moreover, a lag phase was detected in the oxidative-renaturation of reduced W62G lysozyme, as based on observations of the recovery of activity. The simulation of the folding process indicated that intermediates were present at the early stages in the folding of the reduced W62G lysozyme. These results suggest that the presence of the intermediates was derived from the random folding at the early stages in the folding process of reduced W62G lysozyme due to the disruption of the structure of the denatured state. Folding thus appears to have been kinetically delayed by these processes, which then led to the significant aggregation of reduced lysozyme. Moreover, from the analysis of amyloid aggregation of the reduced lysozymes, it was suggested that the disruption of the residual structure in denatured state by W62G mutation deterred the formation of the amyloid fibrils of lysozyme.     

Comparison of the kinetics of S-S bond, secondary structure, and active site formation during refolding of reduced denatured hen egg white lysozyme

To investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics of reoxidation of denatured and reduced lysozyme under the same refolding conditions as those previously used to investigate the kinetics of regain of its circular dichroism (CD), fluorescence, and activity. At different stages of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the various oxidation states present in the samples were identified by electrospray-mass spectrometry. Thus, it was possible to monitor the appearance and/or disappearance of the species with 0 to 4 disulfide bonds. Using a simulation program, these kinetics were compared with those of regain of far-UV CD, fluorescence, and enzymatic activity and were discussed in terms of a refined model for the refolding of reduced hen egg white lysozyme.     

Formation of amyloid fibrils from fully reduced hen egg white lysozyme

The fully reduced hen egg white lysozyme (HEWL), which is a good model of random coil structure, has been converted to highly organized amyloid fibrils at low pH by adding ethanol. In the presence of 90% (v/v) ethanol, the fully reduced HEWL adopts beta-sheet secondary structure at pH 4.5 and 5.0, and an alpha-to-beta transition is observed at pH 4.0. A red shift of the Congo red absorption spectrum caused by the precipitation of the fully reduced HEWL in the presence of 90% (v/v) ethanol is typical of the presence of amyloid aggregation. EM reveals unbranched fibrils with a diameter of 2-5 nm and as long as 1-2 microm. The pH dependence of the initial structure of the fully reduced HEWL in the presence of 90% (v/v) ethanol suggests that Asp and His residues may play an important role.     

Antibody-detected folding: kinetics of surface epitope formation are distinct from other folding phases

The rate of macromolecular surface formation in yeast iso-2 cytochrome c and its site-specific mutant, N52I iso-2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody-detected (ad) kinetic phase accompanying epitope formation has k(ad) = 0.2 s(-1) and is approximately 40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c (k2f approximately 8 s(-1)), but occurs prior to the absorbance- and fluorescence-detected slow folding steps (k1a approximately 0.06 s(-1); k1b approximately 0.09 s(-1)). N5I iso-2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation. A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter. Our results suggest that non-native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso-2 cytochrome c. This study illustrates that surface formation can be coupled to early events in protein folding. Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding.     

Identification and characterization of folding inhibitors of hen egg lysozyme: an example of a new paradigm of drug design

Studies of protein folding indicate the presence of native contacts in the denatured state, giving rise to folding elements which contribute to the accomplishment of the native state. The possibility of finding molecules which can interact with specific folding elements of a target protein preventing it from reaching its native state, and hence from becoming biologically active, is particularly attractive. The notion that folding elements not only provide molecular recognition directing the folding process, but also have conserved sequence, implies that targeting such elements will make protein folding inhibitors less susceptible to mutations which, in many cases, abrogate drug effects. The folding-inhibition strategy can lead to a truly novel and rational approach to drug design, aside from providing new insight into folding. This is illustrated in the case of hen egg lysozyme.     

Cosolvent-assisted oxidative folding of a bicyclic alpha-conotoxin ImI.

alpha-Conotoxin ImI is a 12-amino acid peptide, found in the venom of the marine snail Conus imperialis. This conotoxin is a selective antagonist of alpha7 nicotinic acetylcholine receptors. To produce biologically active alpha-ImI, disulfide bonds must be formed between Cys2-Cys8 and Cys3-Cys12. Oxidative folding of bicyclic conotoxins, such as alpha-ImI, has been traditionally achieved using two-step oxidation protocols with orthogonal protection on two native pairs of cysteines. In this work, two alternative oxidation protocols were explored: (1) the recently described one-pot oxidation of t-butyl/4-methylbenzyl protected Cys pairs and (2) direct oxidative folding. In contrast to the first method, the latter one resulted in high yields of correctly folded alpha-ImI. The addition of organic cosolvents, such as methanol, ethanol or isopropanol into the folding mixture significantly increased the accumulation of the native peptide. This effect was also observed for another conotoxin, alpha-PnIA. It is suggested that cosolvent-assisted direct oxidation might be of general use for other bicyclic alpha-conotoxins, but efficiency should be assessed on a case-by-case basis.     

Structure and heterogeneity of the one- and two-disulfide folding intermediates of tick anticoagulant peptide

Tick anticoagulant peptide (TAP) is a factor Xa-specific inhibitor and is structurally homologous to bovine pancreatic trypsin inhibitor (BPTI). The fully reduced TAP refolds spontaneously to form the native structure under a wide variation of redox buffers. The folding intermediates of TAP consist of at least 22 fractions of one-disulfide, two-disulfide, and three-disulfide scrambled isomers. Three species of well-populated one- and two-disulfide intermediates were isolated and structurally characterized. The predominant one-disulfide species contains TAP-(Cys33—Cys55). Two major two-disulfide isomers were TAP-(Cys33—Cys55, Cys15—Cys39) and TAP-(Cys33—Cys55, Cys5—Cys39). Both Cys33—Cys55 and Cys15—Cys39 are native disulfides of TAP. These three species are structural counterparts of BPTI-(Cys30—Cys51), BPTI-(Cys30—Cys51, Cys14—Cys38), and BPTI-(Cys30—Cys51,Cys5—Cys38), which have been shown to be the major intermediates of BPTI folding. In addition, time-course-trapped folding intermediates of TAP, consisting of about 47% one-disulfide species and 30% two-disulfide species, were collectively digested with thermolysin, and fragmented peptides were analyzed by Edman sequencing and mass spectrometry in order to characterize the disulfide-containing peptides. Among the 15 possible single-disulfide pairings of TAP, 10 (2 native and 8 nonnative) were found as structural components of its one- and two-disulfide folding intermediates. The results demonstrate that the major folding intermediates of TAP bear structural homology to those of BPTI. However, the folding pathway of TAP differs from that of BPTI by (a) a higher degree of heterogeneity of one- and two-disulfide intermediates and (b) the presence of three-disulfide scrambled isomers as folding intermediates. Mechanism(s) that may account for these diversities are proposed and discussed.     

In vitro insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient Ⅰ was formed from P3A through thiol/disulfide exchange reaction; then, transients Ⅱ and Ⅲ, each containing two native disulfides, were formed through the recognition and interaction of transient Ⅰ with P4B or P6B and the thiol group's oxidation reaction mainly using GSSG as oxidative reagent; finally, transients Ⅱ and Ⅲ, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.     

In vitro insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates: two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II and III, each containing two native disulfides, were formed through the recognition and interaction of transient I with P4B or P6B and the thiol group's oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II and III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.     

Characterisation of the dominant oxidative folding intermediate of hen lysozyme.

Reduced denatured lysozyme has been oxidised and refolded at pH values close to neutral in an efficient way by dilution from buffers containing 8.0 M urea, and refolding intermediates were separated by reverse-phase HPLC at pH 2. By using peptic digestion in combination with high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem MS/MS the dominant intermediate was identified to be des-[76-94]. This species has three of the four native disulphide bonds, but lacks the Cys76-Cys94 disulphide bond which connects the two folding domains in the native protein. Characterisation of des-[76-94] by 2D1H NMR shows that it has a highly native-like structure. This provides an explanation for the accumulation of this species during refolding as direct oxidation to the fully native protein will be restricted by the burial of Cys94 in the protein interior.     

Further experimental studies of the disulfide folding transition of ribonuclease A

Two very different mechanisms of folding have been proposed from experimental studies of disulfide formation in reduced ribonuclease A. (1) A pathway in which the rate-limiting step separates fully folded protein from all other disulfide intermediates and occurs solely in three-disulfide intermediates. (2) A multiple pathway mechanism with different rate-limiting steps for each pathway. The various rate-limiting steps involve disulfide breakage, formation, and rearrangement in intermediates with one, two, three, and four protein disulfides. To distinguish between these two mechanisms, we have carried out further studies of both unfolding and refolding. Refolding of reduced ribonuclease A requires three-disulfide intermediates to accumulate; negligible refolding occurs when only the nearly random one- and two-disulfide intermediate species are populated. Therefore, no rate-limiting steps of the type postulated in mechanism (2) occur in intermediates with one and two protein disulfides. Unfolding and disulfide reduction is an all-or-none process; no disulfide intermediates accumulate to detectable levels or precede the rate-limiting step. Mechanism (2) requires that such intermediates precede the rate-limiting step and accumulate to substantial levels. The different proposals were shown not to result from the use of different solution conditions or disulfide reagents; the two sets of data are not inconsistent. Instead, the inappropriate mechanism (2) resulted from an incorrect kinetic analysis and misinterpretation of the kinetics of disulfide formation and breakage.     

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.     

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme.

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.     

Linear correlation between thermal stability and folding kinetics of lysozyme

We have studied the refolding and thermal denaturation of hen egg white lysozyme in a wide range of pH values (from 1.5 to 9.4) using stopped-flow circular dichroism (CD) and differential scanning calorimetry (DSC). A linear correlation was found between the thermal denaturation temperature (T(m)) and the logarithm of the refolding rate of the slow folding phase of hen egg white lysozyme (lnk(2)).     

Subdomain interactions as a determinant in the folding and stability of T4 lysozyme.

The folding of large, multidomain proteins involves the hierarchical assembly of individual domains. It remains unclear whether the stability and folding of small, single-domain proteins occurs through a comparable assembly of small, autonomous folding units. We have investigated the relationship between two subdomains of the protein T4 lysozyme. Thermodynamically, T4 lysozyme behaves as a cooperative unit and the unfolding transition fits a two-state model. The structure of the protein, however, resembles a dumbbell with two potential subdomains: an N-terminal subdomain (residues 13-75), and a C-terminal subdomain (residues 76-164 and 1-12). To investigate the effect of uncoupling these two subdomains within the context of the native protein, we created two circular permutations, both at the subdomain interface (residues 13 and 75). Both variants adopt an active wild-type T4 lysozyme fold. The protein starting with residue 13 is 3 kcal/mol less stable than wild type, whereas the protein beginning at residue 75 is 9 kcal/mol less stable, suggesting that the placement of the termini has a major effect on protein stability while minimally affecting the fold. When isolated as protein fragments, the C-terminal subdomain folds into a marginally stable helical structure, whereas the N-terminal subdomain is predominantly unfolded. ANS fluorescence studies indicate that, at low pH, the C-terminal subdomain adopts a loosely packed acid state. An acid state intermediate is also seen for all of the full-length variants. We propose that this acid state is comprised of an unfolded N-terminal subdomain and a loosely folded C-terminal subdomain.