Effects of disulfide bonds on compactness of protein molecules revealed by volume, compressibility, and expansibility changes during reduction

To elucidate the effects of disulfide bonds on the compactness of protein molecules, the partial specific volume (v(o)) and coefficients of adiabatic compressibility (beta(s)(o)) and thermal expansibility (alpha) of five globular proteins (ovalbumin, beta-lactoglobulin, lysozyme, ribonuclease A, and bovine serum albumin) were measured in aqueous solutions with pH values of 7 and 2 at 25 degrees C when their disulfide bonds were totally reduced by carboxamidomethylation. Circular dichroism and fluorescence spectra show that the secondary and tertiary structures are partly disrupted by reduction, depending on the number of disulfide bonds in the proteins and the pH of the medium. The conformational changes are accompanied by decreases in v(o) and beta(s)(o) and by an increase in alpha, indicating that reduction decreases the internal cavity and increases surface hydration. The beta(s)(o) values of native or oxidized proteins decrease, and the effects of reduction on the volumetric parameters become more significant as the number of disulfide bonds increases and as they are formed over a larger distance in the primary structure. These results demonstrate that disulfide bonds play an important role, mainly via entropic forces, in the three-dimensional structure and compactness of protein molecules.     

Extraction of flexibly structured protein in AOT reverse micelles: the flexible structure of protein is the dominant factor for its incorporation into reverse micelles

The extraction of flexibly-structured protein in Aerosol-OT (AOT)/isooctane reverse micelles was investigated. A flexibly-structured lysozyme was prepared by reduction and carboxymethylation of the disulfide bonds in the lysozyme molecule. For a comparison, lysozymes whose surface hydrophobicity was modified by monoacylation of the amino groups were also used. The extraction rate of the flexibly-structured lysozyme into the micellar phase was greater than that of the native and monoacylated lysozymes, although the free energy change of the lysozyme prepared by breaking the disulfide bonds was smaller than that of the lysozymes whose surfaces were monoacylated. Viscosity measurement of the micellar organic phase containing the modified lysozymes indicated that extraction of the flexibly-structured lysozyme changed the micelle–micelle interaction, while measurement of the interfacial tension between the AOT/isooctane and protein aqueous systems showed the flexibly-structured lysozyme to be the most amphiphilic in character. These results indicated that the flexible structure of a protein was more dominant than its surface hydrophobicity for its incorporation into reverse micelles, and that it leads to greater micelle–micelle interaction.     

Mechanistic insight of photo-induced aggregation of chicken egg white lysozyme: the interplay between hydrophobic interactions and formation of intermolecular disulfide bonds

Recently, it was reported that ultraviolet (UV) illumination could trigger the unfolding of proteins by disrupting the buried disulfide bonds. However, the consequence of such unfolding has not been adequately evaluated. Here, we report that unfolded chicken egg white lysozyme (CEWL) triggered by UV illumination can form uniform globular aggregates as confirmed by dynamic light scattering, atomic force microscopy, and transmission electron microscopy. The assembling process of such aggregates was also monitored by several other methods, such as circular dichroism, fluorescence spectroscopy, mass spectrometry based on chymotrypsin digestion, ANS-binding assay, Ellman essay, and SDS-PAGE. Our finding is that due to the dissociation of the native disulfide bonds by UV illumination, CEWL undergoes drastic conformational changes resulting in the exposure of some hydrophobic residues and free thiols. Subsequently, these partially unfolded molecules self-assemble into small granules driven by intermolecular hydrophobic interaction. With longer UV illumination or longer incubation time, these granules can further self-assemble into larger globular aggregates. The combined effects from both the hydrophobic interaction and the formation of intermolecular disulfide bonds dominate this process. Additionally, similar aggregation behavior can also be found in other three typical disulfide-bonded proteins, that is, α-lactalbumin, RNase A, and bovine serum albumin. Thus, we propose that such aggregation behavior might be a general mechanism for some disulfide-bonded proteins under UV irradiation.     

Aggregate formation and the structure of the aggregates of disulfide-reduced proteins

Aggregate formation and the structure of the aggregates of disulfide-reduced proteins were investigated using -lactalbumin and lysozyme as model proteins. First, reducing conditions were adjusted so that only one of the four disulfide bonds present in each native protein was cleaved. These three-disulfide (3SS) proteins are known to adopt almost native conformations, yet formed precipitates with a basic peptide, lactoferricin, and heparin and heparin fragment, respectively, at concentrations at which native proteins mixed with these compounds remained clear. The 3SS-lysozyme also formed precipitates in the absence of these ligands. Thus, subtle structural changes could lead to aggregation. Electron microscopy revealed fibrillar structures in the aggregates of extensively reduced proteins in the absence of ligands but not in their presence, which shows that the reduction of disulfide bonds suffices for fibril formation and that ligands inhibit fibril formation.     

Beta subunit of (Na+ + K+)-ATPase contains three disulfide bonds

Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., & Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, beta-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native beta subunit: 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the beta subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the beta subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the beta subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the beta subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the beta subunit from the titration experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

The nonconsecutive disulfide bond of Escherichia coli phytase (AppA) renders it dependent on the protein-disulfide isomerase, DsbC

The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane.     

Determination of three disulfide bonds and one free sulfhydryl in the beta subunit of (Na,K)-ATPase

The oxidation state of the 7 cyst(e)ine residues of the beta subunit of lamb kidney (Na,K)-ATPase was determined. The fluorescent sulfhydryl-reactive reagent 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole was utilized before and after reduction of disulfide bonds to determine the number and location of disulfide bonds present in the beta subunit. Treatment of tryptic peptides of the beta subunit separated by reverse phase high performance liquid chromatography with a reducing agent and 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole allowed identification of which residues were disulfide-linked. The results indicated that there is only one free sulfhydryl, Cys44, and that Cys125 is disulfide-bonded to Cys148, Cys158 is disulfide-linked to Cys174, and Cys212 is disulfide-bonded to Cys275. All three disulfide bonds were shown to be reduced in the presence of high concentrations of beta-mercaptoethanol and elevated temperature. These conditions also lead to a loss of (Na,K)-ATPase activity.     

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.     

Analysis of catalytic properties of hen egg white lysozyme during renaturation from denatured and reduced material.

Catalytic properties of hen egg white lysozyme were analyzed during the renaturation of the enzyme from completely reduced and denatured material. The formation of intermediate folding products and the generation of native lysozyme was monitored by acetic acid/urea electrophoresis. The results showed that during the beginning of renaturation almost all reduced and denatured lysozyme is converted to forms possessing lower compactness than native lysozyme, probably as a result of formation of only one or two disulfide bonds. Kinetic analysis of lysozyme during renaturation showed that the generation of lysozyme with four disulfide bonds was not necessarily equivalent to the formation lysozyme with native-like catalytic properties. It appeared that the formation rate of the structures of the structures of the substrate binding site and of the catalytic site were limited by the generation of four disulfide bonds containing lysozyme. The catalytic properties of intermediate folding products made it evident that the final structures of the substrate binding site and of the catalytic site were formed after the generation of all disulfide bonds.     

Oxidative Folding of Conopeptides Modified by <Emphasis Type="Italic">Conus</Emphasis> Protein Disulfide Isomerase

Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis–trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis–trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression.     

P22 tailspike trimer assembly is governed by interchain redox associations

Though disulfide bonds are absent from P22 tailspike protein in its native state, a disulfide-bonded trimeric intermediate has been identified in the tailspike folding and assembly pathway in vitro. The formation of disulfide bonds is critical to efficient assembly of native trimers as mutations at C-terminal cysteines reduce or inhibit trimer formation. We investigated the effect of different redox folding environments on tailspike formation to discover if simple changes in reducing potential would facilitate trimer formation. Expression of tailspike in trxB cell lines with more oxidizing cytoplasms led to lower trimer yields; however, observed assembly rates were unchanged. In vitro, the presence of any redox buffer decreased the overall yield compared to non-redox buffered controls; however, the greatest yields of the native trimer were obtained in reducing rather than oxidizing environments at pH 7. Slightly faster trimer formation rates were observed in the redox samples at pH 7, perhaps by accelerating the reduction of the disulfide-bonded protrimer to the native trimer. These rates and the effects of the redox system were found to depend greatly on the pH of the refolding reaction. Oxidized glutathione (GSSG) trapped a tailspike intermediate, likely as a mixed disulfide. This trapped intermediate was able to form native trimer upon addition of dithiothreitol (DTT), indicating that the trapped intermediate is on the assembly pathway, rather than the aggregation pathway. Thus, the presence of redox agents interfered with the ability of the tailspike monomers to associate, demonstrating that disulfide associations play an important role during the assembly of this cytoplasmic protein.     

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.     

Enzymic and immunochemical properties of lysozyme. X. Conformation, enzymic activity and immunochemistry of lysozyme reduced at two carboxyl groups.

Reduction of lysozyme by diborane, followed by air oxidation of the reduced disulfides and chromatography on CM-cellulose, yielded a homogeneous derivative. In the derivative, the carboxyl groups of aspartic acid 119 and the end-chain leucine residue were reduced to their corresponding alcohols. Correct re-forming of the disulfide bonds was demonstrated by peptide mapping of the tryptic hydrolysates of the derivative and lysozyme without breaking the disulfide bonds, followed by identification of the disulfide-containing peptides. Correct disulfide pairing in the two-disulfide peptide in the tryptic hydrolysate was established from its immunochemical behavior. Preparations of the two-disulfide fragment from lysozyme and derivative had equal inhibitory activities (26 or 32%) of the reaction of lysozyme with two homologous antisera. In ORD measurements, lysozyme and the derivative had equal rotatory powers at neutral pH. However, the bo value for the derivative decreased by about 10%. Below pH 6.4 and above pH 8.0, the derivative was less rotatory than native lysozyme. In CD measurements at neutral pH, the negative ellipticity bands at 220 and 208 nm showed little or no decrease in the derivative relative to the native protein. Although conformational differences between the derivative and its parent protein were almost undetectable by ORD and CD measurements, they were readily detected by chemical monitoring of the conformation. In the derivative, both accessibility to tryptic hydrolysis and reducibility of the disulfide bonds increased markedly. The enzymic activity of the derivative was decreased but retained the same pH optimum. With antisera to lysozyme or antisera to the derivative, lysozyme and its derivative possessed equal antigenic reactivities. The immunochemical findings further confirm the correct refolding of the disulfides. Also, they indicate that aspartic acid 119 and the C-terminal leucine residue are not part of an antigenic reactive region in lysozyme.     

Analysis of the early stage of the folding process of reduced lysozyme using all lysozyme variants containing a pair of cysteines

Twenty-eight hen lysozyme variants that contained a pair of cysteines were constructed to examine the formation of the individual native and nonnative disulfide bonds. We analyzed the extent of the formation of a disulfide bond in each lysozyme variant using a redox buffer (pH 8) containing 1.0 mM reduced and 0.1 mM oxidized glutathione in the absence or presence of 6 M guanidine hydrochloride. In the presence of 6 M guanidine hydrochloride, the extent of the formation of the disulfide bond in each lysozyme variant was proportional to the distance between cysteine residues, indicating that reduced hen lysozyme under a highly denaturing condition adopted a randomly coiled structure. In aqueous solution, the formations of all disulfide bonds occurred much more easily than under a denatured condition. This finding indicated that reduced lysozyme had a somewhat compact structure. Moreover, the scattering data for the extents of the formation of the disulfide bonds among all lysozyme variants were observed. These results suggested that the nonrandom folding occurred in the early stage of the folding of reduced lysozyme, which should provide new insight into the early-stage events in the folding process of reduced lysozyme.     

Cooperative folding of the isolated alpha-helical domain of hen egg-white lysozyme.

Proteins in the alpha-lactalbumin and c-type lysozyme family have been studied extensively as model systems in protein folding. Early formation of the alpha-helical domain is observed in both alpha-lactalbumin and c-type lysozyme; however, the details of the kinetic folding pathways are significantly different. The major folding intermediate of hen egg-white lysozyme has a cooperatively formed tertiary structure, whereas the intermediate of alpha-lactalbumin exhibits the characteristics of a molten globule. In this study, we have designed and constructed an isolated alpha-helical domain of hen egg-white lysozyme, called Lyso-alpha, as a model of the lysozyme folding intermediate that is stable at equilibrium. Disulfide-exchange studies show that under native conditions, the cysteine residues in Lyso-alpha prefer to form the same set of disulfide bonds as in the alpha-helical domain of full-length lysozyme. Under denaturing conditions, formation of the nearest-neighbor disulfide bonds is strongly preferred. In contrast to the isolated alpha-helical domain of alpha-lactalbumin, Lyso-alpha with two native disulfide bonds exhibits a well-defined tertiary structure, as indicated by cooperative thermal unfolding and a well-dispersed NMR spectrum. Thus, the determinants for formation of the cooperative side-chain interactions are located mainly in the alpha-helical domain. Our studies suggest that the difference in kinetic folding pathways between alpha-lactalbumin and lysozyme can be explained by the difference in packing density between secondary structural elements and support the hypothesis that the structured regions in a protein folding intermediate may correspond to regions that can fold independently.     

Disulfide-bonded multimers of proteoglycan 4 (PRG4) are present in normal synovial fluids

Background

The proteoglycan 4 (PRG4) gene encodes for a mucin-like O-linked glycosylated protein with several names, including lubricin and superficial zone protein. The objective of this study was to analyze PRG4 in normal bovine calf and steer synovial fluids for evidence of native multimers formed by intermolecular disulfide bonds.

Methods

A combination of mucin biochemical techniques, with antibodies to both terminal domains and the mucin-like domain of PRG4, were used for analyses.

Results

Multimers were present in both calf and steer fluids, and reduction and alkylation converts the multimeric complex (likely dimeric) into monomeric subunits. Tandem mass spectrometry analyses supported the Western blot data and identified PRG4 in the reduced ∼ 345 kDa monomeric form. Interestingly, ∼ 70 kDa fragments released upon reduction contained peptides from both the N and C terminal regions, which most likely represent fragments of a sparsely glycosylated PRG4 population that are disulfide-linked to extensively glycosylated, intact monomers.

Conclusions

The analyses described here have demonstrated the presence of native disulfide-bonded multimers of PRG4 in normal bovine synovial fluids.

General significance

These structures are similar to those described for multimerization of mucins in general. Such multimerization and proteolytic cleavage of PRG4 may have functional significance in joint health and disease.     

Assignment of the three disulfide bonds of Selenocosmia huwena lectin-I from the venom of spider Selenocosmia huwena.

The positions of the disulfide bonds of Selenocosmia huwena lectin-I (SHL-I) from the venom of the Chinese bird spider S. huwena have been determined. The existence of three disulfide bonds in the native SHL-I was proved by matrix-assisted laser desorption ionization time-of-flight mass spectroscopic analysis. To map the disulfide bonds, native SHL-I was proteolytically digested. The resulting peptides were separated by reverse phase high-performance liquid chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopic analysis indicated the presence of one disulfide bond Cys7-Cys19. The partially reduced peptides by using Tris-(2-carboxyethyl)-phosphine at pH 3.0 were purified by reverse phase high-performance liquid chromatography. Four M Guanidine-HCl was found to increase the yields of partially reduced peptides prominently. The free thiols were carboxamidomethlate by iodoacetamide. The specific location of another disulfide bond Cys2-Cys14 was proved by comparing N-terminal sequencing analysis of the partially reduced and alkylated SHL-I with that of the intact peptide. Finally, the three disulfide linkage of SHL-I could be assigned as Cys2-Cys14, Cys7-Cys19, Cys13-Cys26.     

The biologically active form of the sea urchin egg receptor for sperm is a disulfide-bonded homo-multimer

Since many cell surface receptors exist in their active form as oligomeric complexes, we have investigated the subunit composition of the biologically active sperm receptor in egg plasma membranes from Strongylocentrotus purpuratus. Electrophoretic analysis of the receptor without prior reduction of disulfide bonds revealed that the surface receptor exists in the form of a disulfide-bonded multimer, estimated to be a tetramer. These findings are in excellent agreement with the fact that the NH2-terminus of the extracellular domain of the sperm receptor is rich in cysteine residues. Studies with cross-linking agents of various length and hydrophobicity suggest that no other major protein is tightly associated with the receptor. Given the multimeric structure of the receptor, we investigated the effect of disulfide bond reduction on its biological activity. Because in quantitative bioassays fertilization was found to be inhibited by treatment of eggs with 5 mM dithiothreitol, we undertook more direct studies of the effect of reduction on properties of the receptor. First, we studied the effect of addition of isolated, pure receptor on fertilization. Whereas the non-reduced, native receptor complex inhibited fertilization in a dose- dependent manner, the reduced and alkylated receptor was inactive. Second, we tested the ability of the isolated receptor to mediate binding of acrosome-reacted sperm to polystyrene beads. Whereas beads coated with native receptor bound sperm, those containing reduced and alkylated receptor did not. Thus, these results demonstrate that the biologically active form of the sea urchin sperm receptor consists only of 350 kD subunits and that these must be linked as a multimer via disulfide bonds to produce a complex that is functional in sperm recognition and binding.     

Analysis of polypeptide molecular weights by electrophoresis in urea

Ten proteins of differing disulfide contents and isoionic points were subjected to disc gel electrophoresis in the presence of 8 urea-0.9 acetic acid to evaluate the use of this technique in determining polypeptide molecular weights. Comparison of the electrophoretic mobilities before and after reduction of the proteins' disulfide bonds demonstrated that only after all disulfide bonds were broken, could their molecular weights be estimated with any degree of accuracy. The expression of the electrophoretic mobilities as a function of the proteins' effective hydrodynamic sizes, thereby taking into account the extent of constraint by disulfide bonds, allowed a comparison of disulfide cross-linked and linear forms of the protein polypeptides. The extent to which intrinsic charge affects a protein's electrophoretic mobility was estimated by comparing alpha-lactalbumin and lysozyme, two proteins of identical size but vastly different isoionic points. They exhibited a 20% difference in mobilities. An apparent slow reduction of disulfide bonds was observed to occur when proteins were exposed to reducing agent at low pH in 8 urea.