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.     

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.     

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.     

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

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

Characterization of refolded hen lysozyme variant lacking two outside disulfide bonds

We characterized a refolded hen lysozyme variant containing only two SS-bonds, C64-C80 and C76-C94 (4CAHEL). From CD spectra and its activity, it was found that the refolded 4CAHEL has a structural topology analogous to wild-type lysozyme (WTHEL). Moreover, the refolded 4CAHEL showed no thermal transition, indicating that it had a character like a molten globule.     

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.     

Comprehensive secondary‐structure analysis of disulfide variants of lysozyme by synchrotron‐radiation vacuum‐ultraviolet circular dichroism

To elucidate the effects of specific disulfide bridges (Cys6‐Cys127, Cys30‐Cys115, Cys64‐Cys80, and Cys76‐Cys94) on the secondary structure of hen lysozyme, the vacuum‐ultraviolet circular dichroism (VUVCD) spectra of 13 species of disulfide‐deficient variants in which Cys residues were replaced with Ala or Ser residues were measured down to 170 nm at pH 2.9 and 25°C using a synchrotron‐radiation VUVCD spectrophotometer. Each variant exhibited a VUVCD spectrum characteristic of a considerable amount of residual secondary structures depending on the positions and numbers of deleted disulfide bridges. The contents of α‐helices, β‐strands, turns, and unordered structures were estimated with the SELCON3 program using the VUVCD spectra and PDB data of 31 reference proteins. The numbers of α‐helix and β‐strand segments were also estimated from the VUVCD data. In general, the secondary structures were more effectively stabilized through entropic forces as the number of disulfide bridges increased and as they were formed over larger distances in the primary structure. The structures of three‐disulfide variants were similar to that of the wild type, but other variants exhibited diminished α‐helices with a border between the ordered and disordered structures around the two‐disulfide variants. The sequences of the secondary structures were predicted for all the variants by combining VUVCD data with a neural‐network method. These results revealed the characteristic role of each disulfide bridge in the formation of secondary structures. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

Backbone 1H, 13C, and 15N resonance assignments for lysozyme from bacteriophage lambda

Lysozyme from lambda bacteriophage (λ lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, λ lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. An interesting feature of λ lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes λ lysozyme an interesting system for studies of protein folding. A comparison of the folding properties of λ lysozyme and hen lysozyme will provide important insights into the role that disulfide bonds play in the refolding pathway of the latter protein. Here we report the ¹H, ¹³C and ¹⁵N backbone resonance assignments for λ lysozyme by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for detailed investigation of the refolding pathway using pulse-labelling hydrogen/deuterium exchange experiments monitored by NMR.     

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.     

A highly amyloidogenic region of hen lysozyme

Amyloid fibrils obtained after incubating hen egg-white lysozyme (HEWL) at pH 2.0 and 65 degrees C for extended periods of time have been found to consist predominantly of fragments of the protein corresponding to residues 49-100, 49-101, 53-100 and 53-101, derived largely from the partial acid hydrolysis of Asp-X peptide bonds. These internal fragments of HEWL encompass part of the beta-domain and all the residues forming the C-helix in the native protein, and contain two internal disulfide bridges Cys64-Cys80 and Cys76-Cys94. The complementary protein fragments, including helices A, B and D of the native protein, are not significantly incorporated into the network of fibrils, but remain largely soluble, in agreement with their predicted lower propensities to aggregate. Further analysis of the properties of different regions of HEWL to form amyloid fibrils was carried out by studying fragments produced by limited proteolysis of the protein by pepsin. Here, we show that only fragment 57-107, but not fragment 1-38/108-129, is able to generate well-defined amyloid fibrils under the conditions used. This finding is of particular importance, as the beta-domain and C-helix of the highly homologous human lysozyme have been shown to unfold locally in the amyloidogenic variant D67H, which is associated with the familial cases of systemic amyloidosis linked to lysozyme deposition. The identification of the highly amyloidogenic character of this region of the polypeptide chain provides strong support for the involvement of partially unfolded species in the initiation of the aggregation events that lead to amyloid deposition in clinical disease.     

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.     

Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme

A three-disulfide form of hen egg white lysozyme with Cys⁶ and Cys¹²⁷ blocked by carboxymethyl groups was prepared, purified, and characterized for eventual use in protein folding experiments. Trypsin digestion followed by proline-specific endopeptidase digestion facilitated the unambiguous assignment of the disulfide bond pairings and the modified residues in this derivative. 3SS-lysozyme demonstrated nearly full enzymatic activity at itspH optimum,pH 5.5. The 3SS-lysozyme derivative and unmodified lysozyme were shown to be identical by CD spectroscopy atpH 3.6. Immunochemical binding assays demonstrated that the conformation of lysozyme was perturbed predominantly only locally by breaking and blocking the disulfide bond between Cys⁶ and Cys¹²⁷. Both 3SS-lysozyme and unmodified lysozyme exhibited reversible thermally induced transitions atpH 2.0 but theT _m of 3SS-lysozyme, 18.9°C, was found to be 34° lower than that of native lysozyme under the same conditions. The conformational chemical potential of the denatured form of unmodified lysozyme was determined from the transition curves to be approximately 6.7 kcal/mol higher than that of the denatured form of 3SS-lysozyme, atpH 2.0 and 35°C, if the conformational chemical potential for the folded forms ofboth 3SS-lysozyme and unmodified lysozyme is arbitrarily assumed to be 0.0 kcal/mol. A calculation of the increase in the theoretical loop entropy of denatured 3SS-lysozyme resulting from the cleavage of the Cys⁶-Cys¹²⁷ disulfide bond, however, yielded a value of only 5.4 kcal/mol for the difference in conformational chemical potential. This suggests that, in addition to the entropic component, there is also an enthalpic contribution to the difference in the conformational chemical potential corresponding to approximately 1.3 kcal/mol. Thus, it is concluded that the reduction and blocking of the disulfide bond between Cys⁶ and Cys¹²⁷ destabilizes 3SS-lysozyme relative to unmodified lysozyme predominantly by stabilizing the denatured conformation by increasing its chain entropy.Cornell Biotechnology Army Research Office Predoctoral Fellow, 1986–1989.     

A maurotoxin with constrained standard disulfide bridging: innovative strategy of chemical synthesis,pharmacology, and docking on K+ channels

Maurotoxin (MTX) is a 34-residue toxin that has been isolated initially from the venom of the scorpion Scorpio maurus palmatus. It presents a large number of pharmacological targets, including small conductance Ca2+-activated and voltage-gated K+ channels. Contrary to other toxins of the alpha-KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C1-C5, C2-C6, C3-C4, and C7-C8 (instead of the conventional C1-C5, C2-C6, C3-C7, and C4-C8, herein referred to as Pi1-like) that does not prevent its folding along the classic alpha/beta scaffold of scorpion toxins. Here, we developed an innovative strategy of chemical peptide synthesis to produce an MTX variant (MTXPi1) with a conventional pattern of disulfide bridging without any alteration of the toxin chemical structure. This strategy was used solely to address the impact of half-cystine pairings on MTX structural properties and pharmacology. The data indicate that MTXPi1 displays some marked changes in affinities toward the target K+ channels. Computed docking analyses using molecular models of both MTXPi1 and the various voltage-gated K+ channel subtypes (Shaker B, Kv1.2, and Kv1.3) were found to correlate with MTXPi1 pharmacology. A functional map detailing the interaction between MTXPi1 and Shaker B channel was generated in line with docking experiments.     

Synthesis, 1H NMR structure, and activity of a three-disulfide-bridged maurotoxin analog designed to restore the consensus motif of scorpion toxins

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus. The toxin displays an exceptionally wide range of pharmacological activity since it binds onto small conductance Ca(2+)-activated K(+) channels and also blocks Kv channels (Shaker, Kv1.2 and Kv1.3). MTX possesses 53-68% sequence identity with HsTx1 and Pi1, two other K(+) channel short chain scorpion toxins cross-linked by four disulfide bridges. These three toxins differ from other K(+)/Cl(-)/Na(+) channel scorpion toxins cross-linked by either three or four disulfide bridges by the presence of an extra half-cystine residue in the middle of a consensus sequence generally associated with the formation of an alpha/beta scaffold (an alpha-helix connected to an antiparallel beta-sheet by two disulfide bridges). Because MTX exhibits an uncommon disulfide bridge organization among known scorpion toxins (C1-C5, C2-C6, C3-C4, and C7-C8 instead of C1-C4, C2-C5, and C3-C6 for three-disulfide-bridged toxins or C1-C5, C2-C6, C3-C7, and C4-C8 for four-disulfide-bridged toxins), we designed and chemically synthesized an MTX analog with three instead of four disulfide bridges ([Abu(19),Abu(34)]MTX) and in which the entire consensus motif of scorpion toxins was restored by the substitution of the two half-cystines in positions 19 and 34 (corresponding to C4 and C8) by two isosteric alpha-aminobutyrate (Abu) derivatives. The three-dimensional structure of [Abu(19), Abu(34)]MTX in solution was solved by (1)H NMR. This analog adopts the alpha/beta scaffold with now conventional half-cystine pairings connecting C1-C5, C2-C6, and C3-C7 (with C4 and C8 replaced by Abu derivatives). This novel arrangement in half-cystine pairings that concerns the last disulfide bridge results mainly in a reorientation of the alpha-helix regarding the beta-sheet structure. In vivo, [Abu(19),Abu(34)]MTX remains lethal in mice as assessed by intracerebroventricular injection of the peptide (LD(50) value of 0. 25 microg/mouse). The structural variations are also accompanied by changes in the pharmacological selectivity of the peptide, suggesting that the organization pattern of disulfide bridges should affect the three-dimensional presentation of certain key residues critical to the blockage of K(+) channel subtypes.     

Effects of organic solvents on protein structures: Observation of a structured helical core in hen egg-white lysozyme in aqueous dimethylsulfoxide

A partly folded state of hen egg-white lysozyme has been characterized in 50% DMSO. Low concentrations of DMSO (<10%) have little effect on the overall folded conformation of lysozyme as seen from ¹H NMR chemical shift dispersion. At increasing DMSO concentrations (>10%) a cooperative transition of the structure to a new, partially folded state is observed. This transition is essentially complete by ∼50% DMSO. NMR studies show an overall decrease in chemical shift dispersion with marked broadening of many resonances. A substantial number of backbone and side chain–side chain NOEs suggests the presence of secondary and tertiary interactions in the intermediate state. Tertiary organization of the aromatic residues is also demonstrated by enhanced near-UV circular dichroism and limited exposure of tryptophans as monitored by iodide quenching of fluorescence. The intermediate state exhibits enhanced binding to hydrophobic dyes. Further, the structural transition from this state to a largely unfolded conformation is cooperative. H/D exchange rates of several amide protons and four indole protons of tryptophans (W28, W108, W111, and W123), measured by refolding from 50% DMSO at different time intervals reveal that protection factors are high for the helical domain, whereas NH groups in the triple stranded antiparallel β-sheet domain are largely solvent-exposed. An ordered hydrophobic core in the intermediate state comprising of helix A, helix B, and helix D is consistent with the high protection factors observed. The structured intermediate in 50% DMSO resembles the early kinetic intermediate observed in the refolding of hen egg white lysozyme, as well as a molten globule state of equine lysozyme at low pH. The results demonstrate the potential use of nonaqueous structure perturbing solvents like DMSO to stabilize partially folded conformations of proteins. Proteins 29:492–507, 1997. © 1997 Wiley-Liss, Inc.     

Structure and dynamic properties of the single disulfide-deficient α-amylase inhibitor [C45A/C73A]tendamistat: An NMR study

Covalent linkages such as disulfide bonds are important for the stabilization of proteins. In the present NMR study we compare the structure and the dynamics of the single disulfide-deficient variant C45A/C73A of the α-amylase inhibitor tendamistat and the wild-type protein, which contains two disulfide bonds (C11-C27 and C45-C73). Complete proton assignment was achieved by standard homonuclear 2D techniques for the variant. Chemical shift differences, intra-strand NOE effects and protected amide proton were used to compare the connectivity of the secondary structure elements of variant and wild-type. Dynamic properties of the wild-type protein were studied by ¹³C_α heteronuclear NOE experiments with carbon in natural abundance. ¹⁵N isotope labeling was necessary to obtain the relaxation parameters of the variant, because of sample degradation. The ¹⁵N resonance assignment was achieved by a ¹⁵N 3D-NOESY-HMQC. Removal of the C45-C73 bond by the C45A/C73A mutation has no influence upon the β-barrel structure of tendamistat beside very local changes at the mutation site. The relaxation data revealed only subtle differences between variant and wild-type on a subnanosecond time scale. Only the N-terminus and G62 in the connecting loop between the anti-parallel β-sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and the secretion efficiency of tendamistat. Proteins 33:285–294, 1998. © 1998 Wiley-Liss, Inc.     

Structure of a stabilizing disulfide bridge mutant that closes the active-site cleft of T4 lysozyme.

The engineered disulfide bridge between residues 21 and 142 of phage T4 lysozyme spans the active-site cleft and can be used as a switch to control the activity of the enzyme (Matsumura, M. & Matthews, B.W., 1989, Science 243, 792-794). In the oxidized form the disulfide increases the melting temperature of the protein by 11 degrees C at pH 2. The crystal structure of this mutant lysozyme has been determined in both the reduced and oxidized forms. In the reduced form, the crystal structure of the mutant is shown to be extremely similar to that of wild type. In the oxidized form, however, the formation of the disulfide bridge causes the alpha-carbons of Cys 21 and Cys 142, on opposite sides of the active-site cleft, to move toward each other by 2.5 A. In association with this movement, the amino-terminal domain of the protein undergoes a rigid-body rotation of 5.1 degrees relative to the carboxy-terminal domain. This rotation occurs about an axis passing through the junction of the amino-terminal and carboxy-terminal domains and is also close to the axis that best fits the apparent thermal motion of the amino-terminal domain seen previously in crystals of wild-type lysozyme. Even though the engineered Cys 21-Cys 142 disulfide links together the amino-terminal and carboxy-terminal domains of T4 lysozyme, it does not reduce the apparent mobility of the one domain relative to the other. The pronounced "hinge-bending" mobility of the amino-terminal domain that is suggested by the crystallographic thermal parameters of wild-type lysozyme persists in the oxidized (and reduced) mutant structures. In the immediate vicinity of the introduced disulfide bridge the mutant structure is more mobile (or disordered) than wild type, so much so that the exact conformation of Cys 21 remains obscure. As with the previously described disulfide bridge between residues 9 and 164 of T4 lysozyme (Pjura, P.E., Matsumura, M., Wozniak, J.A., & Matthews, B.W., 1990, Biochemistry 29, 2592-2598), the engineered cross-link substantially enhances the stability of the protein without making the folded structure more rigid.     

Raman optical activity characterization of native and molten globule states of equine lysozyme: comparison with hen lysozyme and bovine alpha-lactalbumin

Vibrational Raman optical activity (ROA) spectra of the calcium-binding lysozyme from equine milk in native and nonnative states are measured and compared with those of the homologous proteins hen egg white lysozyme and bovine alpha-lactalbumin. The ROA spectrum of holo equine lysozyme at pH 4.6 and 22 degrees C closely resembles that of hen lysozyme in regions sensitive to backbone and side chain conformations, indicating similarity of the overall secondary and tertiary structures. However, the intensity of a strong positive ROA band at approximately 1340 cm(-1), which is assigned to a hydrated form of alpha helix, is more similar to that in the ROA spectrum of bovine alpha-lactalbumin than hen lysozyme and may be associated with the greater flexibility and calcium-binding ability of equine lysozyme and bovine alpha-lactalbumin compared with hen lysozyme. In place of a strong sharp positive ROA band at approximately 1300 cm(-1) in hen lysozyme that is assigned to an alpha helix in a more hydrophobic environment, equine lysozyme shows a broader band centered at approximately 1305 cm(-1), which may reflect greater heterogeneity in some alpha-helical sequences. The ROA spectrum of apo equine lysozyme at pH 4.6 and 22 degrees C is almost identical to that of the holo protein, which indicates that loss of calcium has little influence on the backbone and side chain conformations, including the calcium-binding loop. From the similarity of their ROA spectra, the A state at pH 1.9 and both 2 and 22 degrees C and the apo form at pH 4.5 and 48 degrees C, which are partially folded denatured (molten globule or state A) forms of equine lysozyme, have similar structures that the ROA suggests contain much hydrated alpha helix. The A state of equine lysozyme is shown by these results to be more highly ordered than that of bovine alpha-lactalbumin, the ROA spectrum of which has more features characteristic of disordered states. A positive tryptophan ROA band at approximately 1551 cm(-1) in the native holo protein disappears in the A state, which is probably due to the presence of nonnative conformations of the tryptophans associated with a previously identified cluster of hydrophobic residues.