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

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

Unpaired cysteine-54 interferes with the ability of an engineered disulfide to stabilize T4 lysozyme

We have introduced an intramolecular disulfide bond into T4 lysozyme and have shown this molecule to be significantly more stable than the wild-type molecule to irreversible thermal inactivation [Perry, L.J., & Wetzel, R. (1984) Science (Washington, D.C.) 226, 555-557]. Wild-type T4 lysozyme contains two free cysteines, at positions 54 and 97, and no disulfide bonds. By directed mutagenesis of the cloned T4 lysozyme gene, we replaced Ile-3 with Cys. Oxidation in vitro generated an intramolecular disulfide bond; proteolytic mapping showed this bond to connect Cys-3 to Cys-97. While this molecule exhibited substantially more stability against thermal inactivation than wild type, its stability was further enhanced by additional modification with thiol-specific reagents. This and other evidence suggest that at basic pH and elevated temperatures Cys-54 is involved in intermolecular thiol/disulfide interchange with the engineered disulfide, leading to inactive oligomers. Mutagenic replacement of Cys-54 with Thr or Val in the disulfide-cross-linked variant generated lysozymes exhibiting greatly enhanced stability toward irreversible thermal inactivation.     

Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme

Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.     

Kinetic determinism of lysozyme folding at high temperatures

Reduced, disordered hen egg lysozyme rapidly regains enzymic activity in a nonenzymic system previously reported from this laboratory. When such regenerations are carried out at high temperatures (where the native enzyme is unstable), native enzyme is formed as a transient intermediate. Thermal inactivation does not depend on irreversible changes in the protein, as shown by experiments wherein thermally inactivated regenerated lysozyme spontaneously reactivates at 37°. These findings are inconsistent with thermodynamic determinism of protein structure.     

The folding pathway of T4 lysozyme: an on-pathway hidden folding intermediate

T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. In earlier studies, an amide hydrogen/deuterium exchange pulse-labeling experiment detected a stable submillisecond intermediate that accumulates before the rate-limiting transition state. It involves the formation of structures in both the N and C-terminal regions. However, a native-state hydrogen exchange experiment subsequently detected an equilibrium intermediate that only involves the formation of the C-terminal domain. Here, using stopped-flow circular dichroism and fluorescence, amide hydrogen exchange-folding competition, and protein engineering methods, we re-examined the folding pathway of T4-lysozyme. We found no evidence for the existence of a stable folding intermediate before the rate-limiting transition state at neutral pH. In addition, using native-state hydrogen exchange-directed protein engineering, we created a mimic of the equilibrium intermediate. We found that the intermediate mimic folds with the same rate as the wild-type protein, suggesting that the equilibrium intermediate is an on-pathway intermediate that exists after the rate-limiting transition state.     

Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region

We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala49 and Ile70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10 degrees C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala49 and Ile70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.     

Striking stabilization of Arc repressor by an engineered disulfide bond

A solvent-exposed Cys11-Cys11' disulfide bond was designed to link the antiparallel strands of the beta sheet both in the Arc repressor dimer and in a single-chain variant in which the Arc subunits are connected by a 15-residue peptide tether. In both proteins, the presence of the disulfide bond increased the T(m) by approximately 40 degrees C. In the single-chain background, the disulfide bond stabilized Arc by 8.5 kcal/mol relative to the reduced form, a significantly larger degree of stabilization than caused by other engineered disulfides and most natural disulfides. This exceptional stabilization arises from a modest effective concentration of the Cys11-Cys11' disulfide in the native state (71 M) and an anomalously low effective concentration in the denatured state (40 microM). Disulfide cross-linking of the two beta strands in the single-chain Arc background accelerated refolding by a factor of 170 into the sub-microsecond time scale. However, the major energetic effect of the disulfide occurs after the transition state for Arc refolding, slowing unfolding by 200 000-fold.     

Structural analysis of an insect lysozyme exhibiting catalytic efficiency at low temperatures

Bombyx mori lysozyme (BmLZ), from the silkworm, is an insect lysozyme. BmLZ has considerable activity at low temperatures and low activation energies compared with those of hen egg white lysozyme (HEWLZ), according to measurements of the temperature dependencies of relative activity (lytic and glycol chitin) and the estimation of activation energies using the Arrhenius equation. Being so active at low temperatures and low activation energies is characteristic of psychrophilic (cold-adapted) enzymes. The three-dimensional structure of BmLZ has been determined by X-ray crystallography at 2.5 A resolution. The core structure of BmLZ is similar to that of c-type lysozymes. However, BmLZ shows some distinct differences in the two exposed loops and the C-terminal region. A detailed comparison of BmLZ and HEWLZ suggests structural rationalizations for the differences in the catalytic efficiency, stability, and mode of activity between these two lysozymes.     

Impact of an easily reducible disulfide bond on the oxidative folding rate of multi-disulfide-containing proteins.

The burial of native disulfide bonds, formed within stable structure in the regeneration of multi-disulfide-containing proteins from their fully reduced states, is a key step in the folding process, as the burial greatly accelerates the oxidative folding rate of the protein by sequestering the native disulfide bonds from thiol-disulfide exchange reactions. Nevertheless, several proteins retain solvent-exposed disulfide bonds in their native structures. Here, we have examined the impact of an easily reducible native disulfide bond on the oxidative folding rate of a protein. Our studies reveal that the susceptibility of the (40-95) disulfide bond of Y92G bovine pancreatic ribonuclease A (RNase A) to reduction results in a reduced rate of oxidative regeneration, compared with wild-type RNase A. In the native state of RNase A, Tyr 92 lies atop its (40-95) disulfide bond, effectively shielding this bond from the reducing agent, thereby promoting protein oxidative regeneration. Our work sheds light on the unique contribution of a local structural element in promoting the oxidative folding of a multi-disulfide-containing protein.     

Guanidinium derivatives bind preferentially and trigger long-distance conformational changes in an engineered T4 lysozyme

The binding of guanidinium ion has been shown to promote a large-scale translation of a tandemly duplicated helix in an engineered mutant of T4 lysozyme. The guanidinium ion acts as a surrogate for the guanidino group of an arginine side chain. Here we determine whether methyl- and ethylguanidinium provide better mimics. The results show that addition of the hydrophobic moieties to the ligand enhances the binding affinity concomitant with reduction in ligand solubility. Crystallographic analysis confirms that binding of the alternative ligands to the engineered site still drives the large-scale conformational change. Thermal analysis and NMR data show, in comparison to guanidinium, an increase in protein stability and in ligand affinity. This is presumably due to the successive increase in hydrophobicity in going from guanidinium to ethylguanidinium. A fluorescence-based optical method was developed to sense the ligand-triggered helix translation in solution. The results are a first step in the de novo design of a molecular switch that is not related to the normal function of the protein.     

Folding of human lysozyme in vivo by the formation of an alternative disulfide bond.

The mutant h-lysozyme, W64CC65A, with Trp64 and Cys65 replaced by Cys and Ala, respectively, was secreted by yeast and purified. Peptide mapping confirmed that W64CC65A contained a nonnative Cys64-Cys81 bond and three native disulfide bonds. The mutant had 2% of the lytic activity of the wild-type lysozyme. The midpoint concentration of the guanidine hydrochloride denaturation curve, the [D]1/2, was 2.7 M for W64CC65A at pH 3.0 and 25 degrees C, whereas the [D]1/2 for the wild-type h-lysozyme was 2.9 M. These results show that the W64CC65A protein is a compactly folded molecule. Our previous results, using the mutant C81A, indicate that Cys81 is not required for correct folding and activity, whereas Cys65 is indispensable (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 65, 7570-7575). Cys64 substituted for Cys65 in W64CC65A, even though the distance between the alpha-carbons at positions 64 and 81 in the wild-type h-lysozyme is not favorable for forming a disulfide bond. Unlike C81A, the mutant W64CC65/81A, which has the additional substitution of Ala for Cys81, did not fold. These results suggest that the absence of both the Cys64-Cys81 bond and the amino acid residue Trp64 caused the misfolding or destabilization of W64CC65/81A in vivo. It is proposed that the formation of the alternative bond, Cys64-Cys81 is important for the folding of W64CC65A in vivo.     

Effect of disulfide bonds on lysozyme dynamics

The effect of destruction of disulfide bonds on the dynamics of proteins was studied by an example of lysozyme by the methods of molecular dynamics. In lysozyme, in the absence of disulfide bonds, the characteristic times of motions of secondary structure devices increased 3-7 times, whereas the amplitudes of fluctuations of secondary structure devices practically did not vary. In the absence of S-S-bonds, the volume of the molecule decreased approximately by 2%, primarily due to a "cleft" between the major and the small domains of lysozyme. Thus, disulfide bonds not only "glue" the secondary structure devices of the protein but also play a role of "rods", maintaining a certain free volume of the molecule necessary for the realization of its functions.     

The folding pathway of T4 lysozyme: the high-resolution structure and folding of a hidden intermediate

Folding intermediates have been detected and characterized for many proteins. However, their structures at atomic resolution have only been determined for two small single domain proteins: Rd-apocytochrome b(562) and engrailed homeo domain. T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. An early native-state hydrogen exchange experiment identified an intermediate with the C-terminal domain folded and the N-terminal domain unfolded. We have used a native-state hydrogen exchange-directed protein engineering approach to populate this intermediate and demonstrated that it is on the folding pathway and exists after the rate-limiting step. Here, we determined its high-resolution structure and the backbone dynamics by multi-dimensional NMR methods. We also characterized the folding behavior of the intermediate using stopped-flow fluorescence, protein engineering, and native-state hydrogen exchange. Unlike the folding intermediates of the two single-domain proteins, which have many non-native side-chain interactions, the structure of the hidden folding intermediate of T4 lysozyme is largely native-like. It folds like many small single domain proteins. These results have implications for understanding the folding mechanism and evolution of multi-domain proteins.     

Evidence for difference in the roles of two cysteine residues involved in disulfide bond formation in the folding of human lysozyme

Human lysozyme is made up of 130 amino acid residues and has four disulfide bonds at Cys6-Cys128, Cys30-Cys116, Cys65-Cys81, and Cys77-Cys95. Our previous results using the Saccharomyces cerevisiae secretion system indicate that the individual disulfide bonds of human lysozyme have different functions in the correct in vivo folding and enzymatic activity of the protein (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). In this paper, we report the results of experiments that were focused on the roles of Cys65 and Cys81 in the folding of human lysozyme protein in yeast. A mutant protein (C81A), in which Cys81 was replaced with Ala, had almost the same enzymatic activity and conformation as those of the native enzyme. On the other hand, another mutant (C65A), in which Cys65 was replaced with Ala, was not found to fold correctly. These results indicate that Cys81 is not a requisite for both correct folding and activity, whereas Cys65 is indispensable. The mutant protein C81A is seen to contain a new, non-native disulfide bond at Cys65-Cys77. The possible occurrence of disulfide bond interchange during our mapping experiments cannot be ruled out by the experimental techniques presently available, but characterization of other mutant proteins and computer analysis suggest that the intramolecular exchange of disulfide bonds is present in the folding pathway of human lysozyme in vivo.     

Stabilities of disulfide bond intermediates in the folding of apamin.

Apamin is an 18-residue bee venom peptide with the sequence CNCKAPETALCARRCQQH-amide and contains 2 disulfide bonds connecting C-1 to C-11 and C-3 to C-15. In the folding of reduced, unfolded apamin to native apamin with two disulfide bonds, the one-disulfide folding intermediate states are not populated to significant levels. To study the properties of the one-disulfide intermediates, we have synthesized two peptide models to mimic the one-disulfide intermediates, Apa-1 and Apa-2, in which two cysteines in the sequence have been replaced by alanines. These peptides can form only one of the native disulfide bonds, C-1 to C-11 in the case of Apa-1 and C-3 to C-15 in the case of Apa-2. The stabilities of these disulfide bonds have been measured as a function of pH, concentration of urea, and temperature, in order to understand which contributions stabilize the disulfide-bonded structures. Using oxidized and reduced glutathione, the equilibrium constants for forming the disulfide bonds at 25 degrees C and pH 7.0 are 0.018 M for Apa-1 and 0.033 M for Apa-2 and show little dependence on pH or temperature. Both disulfide bonds are destabilized slightly (by approximately a factor of 2) between 0 and 8 M urea. Circular dichroism spectra indicate that although both Apa-1 and Apa-2 exhibit some structure, Apa-2 exhibits more than Apa-1. The results suggest that in the folding of apamin, the one-disulfide intermediate containing the C-3 to C-15 disulfide bond, as in Apa-2, is favored slightly. Secondary structure provides modest stabilization to this intermediate.     

Oxidative folding of Amaranthus alpha-amylase inhibitor: disulfide bond formation and conformational folding

Oxidative folding is the fusion of native disulfide bond formation with conformational folding. This complex process is guided by two types of interactions: first, covalent interactions between cysteine residues, which transform into native disulfide bridges, and second, non-covalent interactions giving rise to secondary and tertiary protein structure. The aim of this work is to understand both types of interactions in the oxidative folding of Amaranthus alpha-amylase inhibitor (AAI) by providing information both at the level of individual disulfide species and at the level of amino acid residue conformation. The cystine-knot disulfides of AAI protein are stabilized in an interdependent manner, and the oxidative folding is characterized by a high heterogeneity of one-, two-, and three-disulfide intermediates. The formation of the most abundant species, the main folding intermediate, is favored over other species even in the absence of non-covalent sequential preferences. Time-resolved NMR and photochemically induced dynamic nuclear polarization spectroscopies were used to follow the oxidative folding at the level of amino acid residue conformation. Because this is the first time that a complete oxidative folding process has been monitored with these two techniques, their results were compared with those obtained at the level of an individual disulfide species. The techniques proved to be valuable for the study of conformational developments and aromatic accessibility changes along oxidative folding pathways. A detailed picture of the oxidative folding of AAI provides a model study that combines different biochemical and biophysical techniques for a fuller understanding of a complex process.     

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.     

Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond

To find a disulfide pair that could stabilize the enzyme human carbonic anhydrase II (HCA II), we grafted the disulfide bridge from the related and unusually stable carbonic anhydrase form from Neisseria gonorrhoeae (NGCA) into the human enzyme. Thus, the two Cys residues at positions 23 and 203 were engineered into a pseudo-wild-type form of HCA II (C206S), giving the mutant C206S/A23C/L203C. The disulfide bond was not formed spontaneously. The native state of the reduced form of the mutant was markedly destabilized (2.9 kcal/mol) compared to that of HCA II. Formation of a disulfide bridge was achieved by treatment by oxidized glutathione. This led to a significant stabilization of the native conformation. Compared to HCA II the unfolding midpoint for the variant was increased from 0.9 to 1.7 M guanidine HCl, corresponding to a stabilization of 3.7 kcal/mol. This makes the human enzyme almost as stable as the model protein NGCA, for which the unfolding of the native state has a midpoint at 2.1 M guanidine HCl. The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition, as judged from intrinsic Trp fluorescence measurements. A molten-globule intermediate is nevertheless formed but is suppressed because of the high denaturant pressure it faces upon rupture of the native state.     

Accelerated secretion of human lysozyme with a disulfide bond mutation.

The mutant human lysozyme, [Ala77, Ala95]lysozyme, in which the disulfide bond Cys77-Cys95 is eliminated, is known to exhibit increased secretion in yeast, compared to wild-type human lysozyme [Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M. & Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967]. To investigate this phenomenon, mammalian cells were used to analyze the secretion kinetics of [Ala77, Ala95]lysozyme and wild-type human lysozyme. The secretion rate of [Ala77, Ala95]lysozyme during the 150-min chase period was significantly accelerated [half-life (t1/2) = 29 min] compared to that of wild-type human lysozyme (t1/2 = 83 min), when expressed at the same levels within the cells. In contrast, after the 150-min chase, the rates of disappearance of both wild-type and mutant human lysozymes within the cells were similar, and considerably slower (t1/2 = 220 min), respectively. The remaining intracellular wild-type human lysozyme was localized mainly in the endoplasmic reticulum, whereas accelerated transport of the [Ala77, Ala95]lysozyme mutant protein from the endoplasmic reticulum to the Golgi apparatus was observed. Also in yeast cells, similar secretion kinetics and the differences in t1/2 for wild-type and mutant human lysozymes during the early chase period were observed. The two-phase kinetics of disappearance of intracellular human lysozymes suggest that only a proportion of the proteins becomes secretion competent soon after synthesis and is completely secreted during the early chase period, whereas others enter the distinct, slow pathways of intracellular transport and/or degradation. Increased secretion of [Ala77, Ala95]lysozyme is possibly due to enhanced competence for secretion acquired in the endoplasmic reticulum at the early stage of transport events, which is closely connected with the removal of a disulfide bond.     

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.