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.     

The transition state in the folding-unfolding reaction of four species of three-disulfide variant of hen lysozyme: the role of each disulfide bridge

The effects of lacking a specific disulfide bridge on the transition state in folding were examined in order to explore the folding-unfolding mechanism of lysozyme. Four species of three-disulfide variant of hen lysozyme (3SS-lysozyme) were prepared by replacing two Cys residues with Ala or Ser: C6S/C127A, C30A/C115A, C64A/C80A and C76A/C94A. The recombinant hen lysozyme was studied as the standard reference containing four authentic disulfide bridges and the extra N-terminal Met: the recombinant hen lysozyme containing the extra N-terminal. Folding rates were measured by monitoring the change in fluorescence intensity associated with tri-N-acetyl-d-glucosamine binding to the active site of refolded lysozyme. It was confirmed that the folding rate of the recombinant hen lysozyme containing the extra N-terminal was the same as that of wild-type lysozyme, and that the folding rate was little affected by the presence of tri-N-acetyl-d-glucosamine (triNAG). The folding rate of C64A/C80A was found to be the fastest and almost the same as that of the recombinant hen lysozyme containing the extra N-terminal, and that of C30A/C115A the second, and that of C6S/C127A the third. The folding rate of C76A/C94A was particularly slow. On the other hand, the unfolding rates which were measured in the presence of triNAG showed the dependence on the concentration of triNAG. The intrinsic unfolding rate in the absence of triNAG was determined by extrapolation. Also in the unfolding rate, C76A/C94A was markedly slower than the others. It was found from the analysis of binding constants of triNAG to C64A/C80A during the unfolding process that the active site of C64A/C80A partly unfolds already prior to the unfolding transition. On the basis of these kinetic data, we suggest that C64A/C80A folding transition can occur with leaving the loop region around SS3 (C64-C80) flexible, while cross-linking by SS4 (C76-C94) is important for the promotion of folding, because it is an indispensable constraint on the way towards the folding transition state.     

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.     

Glycerol-enhanced detection of a preferential structure latent in unstructured 1SS-variants of lysozyme

Four species of 1SS-varinats of lysozyme were almost unstructured in water, judged from their near-UV CD and (1) H-(15) N-HSQC spectra. Some preferential structure might exist in such a disordered state, but the population of molecules in such a conformation must have been too small to be detected by spectroscopic methods. Indeed, our previous study showed that the addition of 30% glycerol induced the unstructured 2SS-variant of lysozyme to form a native-like structure. To extend this method to more disordered proteins, we attempted to detect some preferential structure latent in unstructured 1SS-variants by the glycerol-enhanced detection. Only in one molecular species of the four 1SS-variants, 1SS[6-127] containing a single disulfide bridge of Cys6-Cys127, a preferential structure was found in the presence of 50% glycerol. It was detected by near-UV CD measurements and the H/D exchange method combined with the NMR spectroscopy. The glycerol-induced structure in 1SS[6-127] was not localized only in the vicinity of Cys6-Cys127, and largely protected regions distributed themselves among A-, B-, and C-helices and Ile55 and Leu56. It was similar to the glycerol-induced structure in 2SS[6-127, 64-80] containing two disulfide bridges of Cys6-Cys127 and Cys64-Cys80, although the former was less rigid than the latter. The role of A-helix (residues 4-15) is proposed as an origin of excellent potential of Cys6-Cys127 for inducing a tertiary structure in the α-domain.     

Two-State Folding of Lysozyme Versus Multiple-State Folding of α-Lactalbumin Illustrated by the Technique of Disulfide Scrambling

The folding of lysozyme and of alpha-lactalbumin exhibits vastly different kinetics and pathways. Existing evidence indicates that folding intermediates of alphaLA form a well-populated equilibrium molten globule state that is absent in the case of hen lysozyme. We demonstrate here such divergent folding mechanisms of lysozyme and alphaLA using the technique of disulfide scrambling. Two extensively unfolded homologous isomers (beads-form) of lysozyme (Cys6-Cys30, Cys64-Cys76, Cys80-Cys94, Cys115-Cys127) and alphaLA (Cys6-Cys28, Cys61-Cys73, Cys77-Cys91, Cys111-Cys120) were allowed to refold in parallel to form the native protein. Folding kinetics was measured by the recovery of the native structure. Folding intermediates, which illustrate the folding pathway, were trapped by quenching disulfide shuffling and were analyzed by reversed-phase high-pressure liquid chromatography. The results revealed that under identical folding conditions, the folding rate of lysozyme is about 30-fold faster than that of alphaLA. Folding intermediates of lysozyme are far less heterogeneous and sparsely populated than those of alphaLA. Numerous predominant on-pathway and off-pathway intermediates observed along the folding pathway of alphaLA are conspicuously absent in the case of lysozyme. The difference is most striking under fast folding conditions performed in the presence of protein disulfide isomerase. Under these conditions, folding of lysozyme undergoes a near two-state mechanism without accumulation of stable folding intermediates.     

The oxidative refolding of hen lysozyme and its catalysis by protein disulfide isomerase.

The oxidative refolding of hen lysozyme has been studied by a variety of time-resolved biophysical methods in conjunction with analysis of folding intermediates using reverse-phase HPLC. In order to achieve this, refolding conditions were designed to reduce aggregation during the early stages of the folding reaction. A complex ensemble of relatively unstructured intermediates with on average two disulfide bonds is formed rapidly from the fully reduced protein after initiation of folding. Following structural collapse, the majority of molecules slowly form the four-disulfide-containing fully native protein via rearrangement of a highly native-like, kinetically trapped intermediate, des-[76-94], although a significant population (approximately 30%) appears to fold more quickly via other three-disulfide intermediates. The folding catalyst PDI increases dramatically both yields and rates of lysozyme refolding, largely by facilitating the conversion of des-[76-94] to the native state. This suggests that acceleration of the folding rate may be an important factor in avoiding aggregation in the intracellular environment.     

NMR characterization of three-disulfide variants of lysozyme, C64A/C80A, C76A/C94A, and C30A/C115A--a marginally stable state in folded proteins

Our earlier NMR study showed that a two-disulfide variant of hen lysozyme containing intra-alpha-domain disulfide bridges, C6-C127 and C30-C115, is partially folded, with the alpha domain tightly folded to the nativelike conformation and the beta domain flexible or unfolded. With a view that the formation of a third disulfide bridge is a key for the accomplishment of the overall chain fold, three-dimensional structures of three-disulfide variants of hen lysozyme lacking one disulfide bridge (C64A/C80A, C76A/C94A, and C30A/C115A) were studied in detail using NMR spectroscopy. Amide hydrogen exchange rates were measured to estimate the degree of conformational fluctuation in a residue-specific manner. The structure of C76A/C94A was found to be quite similar to that of the wild type, except for the peptide segment of residues 74-78. The structure of C64A/C80A was considerably disordered in the entire region of the loop (residues 62-79). Further, it was found that a network of hydrogen bonds within the beta sheet and the 3(10) helix in the beta domain were disrupted and fluctuating. In C30A/C115A, the D helix was unstructured and the interface of the B helix with the D helix was significantly perturbed. However, the structural disorder generated in the hydrophobic core of the alpha domain was prevented by the C helix from propagating toward the beta domain. A marginally stable state in folded proteins is discussed based on the structures remaining in each three-disulfide variant.     

A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure of native lysozyme are operational even when one of these three native disulfide bonds between Cys 6 and Cys 127, Cys 76 and Cys 94, and Cys 64 and 80 is open.     

Evidence for an initiation site for hen lysozyme folding from the reduced form using its dissected peptide fragments.

We prepared two dissected fragments of hen lysozyme and examined whether or not these two fragments associated to form a native-like structure. One (Fragment I) is the peptide fragment Asn59-homoserine-105 containing Cys64-Cys80 and Cys76-Cys94. The other (Fragment II) is the peptide fragment Lys1-homoserine-58 connected by two disulfide bridges, Cys6-Cys127 and Cys30-Cys115, to the peptide fragment Asn106-Leu129. It was found that the Fragment I immobilized in the cuvette formed an equimolar complex with Fragment II (K(d) = 3.3x10(-4) M at pH 8 and 25 degrees C) by means of surface plasmon resonance. Moreover, from analyses by circular dichroism spectroscopy and ion-exchange chromatography of the mixture of Fragments I and II at pH 8 under non-reducing conditions, it was suggested that these fragments associated to give the native-like structure. However, the mutant Fragment I in which Cys64-Cys80 and Cys76-Cys94 are lacking owing to the mutation of Cys to Ala, or the mutant fragment in which Trp62 is mutated to Gly, did not form the native-like species with Fragment II, because the mutant Fragment I derived from mutant lysozymes had no local conformation due to mutations. Considering our previous results where the preferential oxidation of two inside disulfide bonds, Cys64-Cys80 and Cys76-Cys94, occurred in the refolding of the fully reduced Fragment I, we suggest that the peptide region corresponding to Fragment I is an initiation site for hen lysozyme folding.     

Glycerol‐induced folding of unstructured disulfide‐deficient lysozyme into a native‐like conformation

2SS[6‐127,64‐80] variant of lysozyme which has two disulfide bridges, Cys6‐Cys127 and Cys64‐Cys80, and lacks the other two disulfide bridges, Cys30‐Cys115 and Cys76‐Cys94, was quite unstructured in water, but a part of the polypeptide chain was gradually frozen into a native‐like conformation with increasing glycerol concentration. It was monitored from the protection factors of amide hydrogens against H/D exchange. In solution containing various concentrations of glycerol, H/D exchange reactions were carried out at pH* 3.0 and 4°C. Then, ¹H‐¹⁵N‐HSQC spectra of partially deuterated protein were measured in a quenching buffer for H/D exchange (95% DMSO/5% D₂O mixture at pH* 5.5 adjusted with dichloroacetate). In a solution of 10% glycerol, the protection factors were nearly equal to 10 at most of residues. With increasing glycerol concentration, some selected regions were further protected, and their protection factors reached about a 1000 in 30% glycerol solution. The highly protected residues were included in A‐, B‐, and C‐helices and β3‐strand, and especially centered on Ile 55 and Leu 56. In 2SS[6‐127,64‐80], long‐range interactions were recovered due to the preferential hydration by glycerol in the hydrophobic box of the α‐domain. Glycerol‐induced recovering of the native‐like structure is discussed from the viewpoint of molten globules growing with the protein folding. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 665–675, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

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.     

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.     

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.     

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.     

Energetics of structural domains in alpha-lactalbumin.

alpha-Lactalbumin is a small, globular protein that is stabilized by four disulfide bonds and contains two structural domains. One of these domains is rich in alpha-helix (the alpha-domain) and has Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds. The other domain is rich in beta-sheet (the beta-domain), has Cys 61-Cys 77 and Cys 73-Cys 91 disulfide bonds, and includes one calcium binding site. To investigate the interaction between domains, we studied derivatives of bovine alpha-lactalbumin differing in the number of disulfide bonds, using calorimetry and CD at different temperatures and solvent conditions. The three-disulfide form, having a reduced Cys 6-Cys 120 disulfide bond with carboxymethylated cysteines, is similar to intact alpha-lactalbumin in secondary and tertiary structure as judged by its ellipticity in the near and far UV. the two-disulfide form of alpha-lactalbumin, having reduced Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds with carboxymethylated cysteines, retains about half the secondary and tertiary structure of the intact alpha-lactalbumin. The remaining structure is able to bind calcium and unfolds cooperatively upon heating, although at lower temperature and with significantly lower enthalpy and entropy. We conclude that, in the two disulfide form, alpha-lactalbumin retains its calcium-binding beta-domain, whereas the alpha-domain is unfolded. It appears that the beta-domain does not require alpha-domain to fold, but its structure is stabilized significantly by the presence of the adjacent folded alpha-domain.     

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.     

Defect in modification at the anticodon wobble nucleotide of mitochondrial tRNA(Lys) with the MERRF encephalomyopathy pathogenic mutation

Hen lysozyme single-disulfide variants were constructed to characterize the structures associated with the formation of individual native disulfide bonds. Circular dichroism spectra and the effective concentration of protein thiol groups showed that the propensity for structure formation was relatively high for Cys-6–Cys-127 and Cys-30–Cys-115 disulfides. The urea concentration dependence of individual effective concentrations showed that the apparent sizes of the structures were 14–50% of the whole molecule. The intrinsic stability of each submolecular structure in a reduced form of protein, obtained by subtracting the entropic contribution of cross-linking, was highest for Cys-64–Cys-80 and lowest for Cys-76–Cys-94 disulfide bonds.     

Analyses of Native Disulfide Bond Formations in the Early Stage of the Folding Process in Mutant Lysozymes Where the Long-Range Interactions in the Denatured State Were Modulated

In order to clarify whether modulation of long-range interactions in the denatured state affect native disulfide bond (SS bond) formations of hen egg white lysozyme (HEL) containing a pair of cysteine residues, we examined the extent of SS bond formation among 12 variants containing a pair of cysteines. The loss of clusters 5 and 6 in the denatured state affected the formation of Cys30-Cys115 and Cys6-Cys127 respectively.