Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein

Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike.     

Characterization of the protrimer intermediate in the folding pathway of the interdigitated beta-helix tailspike protein

P22 tailspike is a homotrimeric, thermostable adhesin that recognizes the O-antigen lipopolysaccharide of Salmonella typhimurium. The 70 kDa subunits include long beta-helix domains. After residue 540, the polypeptide chains change their path and wrap around one another, with extensive interchain contacts. Formation of this interdigitated domain intimately couples the chain folding and assembly mechanisms. The earliest detectable trimeric intermediate in the tailspike folding and assembly pathway is the protrimer, suspected to be a precursor of the native trimer structure. We have directly analyzed the kinetics of in vitro protrimer formation and disappearance for wild type and mutant tailspike proteins. The results confirm that the protrimer intermediate is an on-pathway intermediate for tailspike folding. Protrimer was originally resolved during tailspike folding because its migration through nondenaturing polyacrylamide gels was significantly retarded with respect to the migration of the native tailspike trimer. By comparing protein mobility versus acrylamide concentration, we find that the retarded mobility of the protrimer is due exclusively to a larger overall size than the native trimer, rather than an altered net surface charge. Experiments with mutant tailspike proteins indicate that the conformation difference between protrimer and native tailspike trimer is localized toward the C-termini of the tailspike polypeptide chains. These results suggest that the transformation of the protrimer to the native tailspike trimer represents the C-terminal interdigitation of the three polypeptide chains. This late step may confer the detergent-resistance, protease-resistance, and thermostability of the native trimer.     

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.     

Three amino acids that are critical to formation and stability of the P22 tailspike trimer

The P22 tailspike protein folds by forming a folding competent monomer species that forms a dimeric, then a non-native trimeric (protrimer) species by addition of folding competent monomers. We have found three residues, R549, R563, and D572, which play a critical role in both the stability of the native tailspike protein and assembly and maturation of the protrimer. King and colleagues reported previously that substitution of R563 to glutamine inhibited protrimer formation. We now show that the R549Q and R563K variants significantly delay the protrimer-to-trimer transition both in vivo and in vitro. Previously, variants that destabilize intermediates have shown wild-type chemical stability. Interestingly, both the R549Q and R563K variants destabilize the tailspike trimer in guanidine denaturation studies, indicating that they represent a new class of tailspike folding variants. R549Q has a midpoint of unfolding at 3.2M guanidine, compared to 5.6M for the wild-type tailspike protein, while R563K has a midpoint of unfolding of 1.8 M. R549Q and R563K also denature over a broader pH range than the wild-type tailspike protein and both proteins have increased sensitivity to pH during refolding, suggesting that both residues are involved in ionic interactions. Our model is that R563 and D572 interact to stabilize the adjacent turn, aiding the assembly of the dimer and protrimer species. We believe that the interaction between R563 and D572 is also critical following assembly of the protrimer to properly orient D572 in order to form a salt bridge with R549 during protrimer maturation.     

Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS

Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS-gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers.     

Stalled folding mutants in the triple beta-helix domain of the phage P22 tailspike adhesin

The trimeric bacteriophage P22 tailspike adhesin exhibits a domain in which three extended strands intertwine, forming a single turn of a triple beta-helix. This domain contains a single hydrophobic core composed of residues contributed by each of the three sister polypeptide chains. The triple beta-helix functions as a molecular clamp, increasing the stability of this elongated structural protein. During folding of the tailspike protein, the last precursor before the native state is a partially folded trimeric intermediate called the protrimer. The transition from the protrimer to the native state results in a structure that is resistant to denaturation by heat, chemical denaturants, and proteases. Random mutations were made in the region encoding residues 540-548, where the sister chains begin to wrap around each other. From a set of 26 unique single amino acid substitutions, we characterized mutations at G546, N547, and I548 that retarded or blocked the protrimer to native trimer transition. In contrast, many non-conservative substitutions were tolerated at residues 540-544. Sucrose gradient analysis showed that protrimer-like mutants had reduced sedimentation, 8.0 S to 8.3 S versus 9.3 S for the native trimer. Mutants affected in the protrimer to native trimer transition were also destabilized in their native state. These data suggest that the folding of the triple beta-helix domain drives transition of the protrimer to the native state and is accompanied by a major rearrangement of polypeptide chains.     

Distinct cysteine sulfhydryl environments detected by analysis of Raman S-hh markers of Cys-->Ser mutant proteins

Very little is known about the character or functional relevance of hydrogen-bonded cysteine sulfhydryl (S-H) groups in proteins. The Raman S-H band is a unique and sensitive probe of the local S-H environment. Here, we report the use of Raman spectroscopy combined with site-specific mutagenesis to document the existence of five distinguishable hydrogen-bonded states of buried cysteine sulfhydryl groups in a native protein. The 666 residue subunit of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteine residues distributed through the elongated structure. The tailspike cysteine residues display an unusual Raman S-H band complex (2500-2600 cm(-1) interval) indicative of diverse S-H hydrogen-bonding interactions in the native trimeric structure. To resolve specific Cys contributions to the complex Raman band we characterized a set of tailspike proteins with each cysteine replaced by a serine. The mutant proteins, once folded, were structurally and functionally indistinguishable from wild-type tailspikes, except for their Raman S-H signatures. Comparison of the Raman spectra of the mutant and wild-type proteins reveals the following hydrogen-bond classes for cysteine sulfhydryl groups. (i) Cys613 forms the strongest S-H...X bond of the tailspike, stronger than any heretofore observed for a protein. (ii) Cys267, Cys287 and Cys458 form robust S-H...X bonds. (iii) Moderate S-H...X bonding occurs for Cys169 and Cys635. (iv) Cys290 and Cys496 form weak hydrogen bonds. (v) It is remarkable that Cys287 contributes two Raman S-H markers, indicating the population of two distinct hydrogen-bonding states. The sum of the S-H Raman signatures of all eight mutants accurately reproduces the composite Raman band of the wild-type tailspike. The diverse cysteine states may be an outcome of the folding and assembly pathway of the tailspike, which though lacking disulfide bonds in the native state, utilizes transient disulfide bonds in the maturation pathway. This Raman study represents the first detailed assessment of local S-H hydrogen bonding in a native protein and provides information not obtainable directly by other structural probes. The method employed here should be applicable to a wide range of cysteine-containing proteins.     

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly.     

The interdigitated beta-helix domain of the P22 tailspike protein acts as a molecular clamp in trimer stabilization

The P22 tailspike adhesin is an elongated thermostable trimer resistant to protease digestion and to denaturation in sodium dodecyl sulfate. Monomeric, dimeric, and protrimeric folding and assembly intermediates lack this stability and are thermolabile. In the native trimer, three right-handed parallel beta-helices (residues 143-540), pack side-by-side around the three-fold axis. After residue 540, these single chain beta-helices terminate and residues 541-567 of the three polypeptide chains wrap around each other to form a three-stranded interdigitated beta-helix. Three mutants located in this region -- G546D, R563Q, and A575T -- blocked formation of native tailspike trimers, and accumulated soluble forms of the mutant polypeptide chains within cells. The substitutions R563Q and A575T appeared to prevent stable association of partially folded monomers. G546D, in the interdigitated region of the chain, blocked tailspike folding at the transition from the partially-folded protrimer to the native trimer. The protrimer-like species accumulating in the G546D mutant melted out at 42 degrees C and was trypsin and SDS sensitive. The G546D defect was not corrected by introduction of global suppressor mutations, which correct kinetic defects in beta-helix folding. The simplest interpretation of these results is that the very high thermostability (T(m) = 88 degrees C), protease and detergent resistance of the native tailspike acquired in the protrimer-to-trimer transition, depends on the formation of the three-stranded interdigitated region. This interdigitated beta-helix appears to function as a molecular clamp insuring thermostable subunit association in the native trimer.     

C-terminal hydrophobic interactions play a critical role in oligomeric assembly of the P22 tailspike trimer

The tailspike protein from the bacteriophage P22 is a well characterized model system for folding and assembly of multimeric proteins. Folding intermediates from both the in vivo and in vitro pathways have been identified, and both the initial folding steps and the protrimer-to-trimer transition have been well studied. In contrast, there has been little experimental evidence to describe the assembly of the protrimer. Previous results indicated that the C terminus plays a critical role in the overall stability of the P22 tailspike protein. Here, we present evidence that the C terminus is also the critical assembly point for trimer assembly. Three truncations of the full-length tailspike protein, TSPΔN, TSPΔC, and TSPΔNC, were generated and tested for their ability to form mixed trimer species. TSPΔN forms mixed trimers with full-length P22 tailspike, but TSPΔC and TSPΔNC are incapable of forming similar mixed trimer species. In addition, mutations in the hydrophobic core of the C terminus were unable to form trimer in vivo. Finally, the hydrophobic-binding dye ANS inhibits the formation of trimer by inhibiting progression through the folding pathway. Taken together, these results suggest that hydrophobic interactions between C-terminal regions of P22 tailspike monomers play a critical role in the assembly of the P22 tailspike trimer.     

Genetic and biochemical analysis of in vivo protein folding and subunit assembly

The in vivo pathway of folding and subunit assembly of a trimeric bacteriophage protein has been studied by characterizing precursors to the native protein and by analyzing temperature-sensitive mutations that kinetically block the pathway. The native trimer is formed via an intermediate composed of three partially folded chains, the protrimer. At 39°C, temperature-sensitive mutations prevent the formation of both the native trimer and the protrimer, possibly by destabilizing earlier intermediates. However, the mutations do not affect the stability of the native protein, formed at 30°C. Thus, these mutations identify amino acid residues involved in interactions that determine the folding pathway.     

Intragenic Suppression of a Capsid Assembly-Defective P22 Tailspike Mutation

The tailspike protein of bacteriophage P22 assembles with mature capsids during the final reaction in phage morphogenesis. The gene 9 mutation hmH3034 synthesizes a tailspike protein with a change at amino acid 100 from Asp to Asn. This mutant form of trimeric tailspike protein fails to assemble with capsids in vivo. By using in vitro quantitative tailspike-capsid assembly assays, this mutant tailspike trimer can be shown to assemble with capsids at very high tailspike concentrations. From these assays, we estimate that this single missense mutation decreases by 100-500-fold the affinity of the tailspike for capsids. Furthermore, hmH3034 tailspike protein has a structural defect which makes the mature tailspike trimers sensitive to SDS at room temperature and causes the trimers to "partially unfold." Spontaneously arising intragenic suppressors of the capsid assembly defect have been isolated. All of these suppressors are changes at amino acid 13 of the tailspike protein, which substitute His, Leu or Ser for the wild type amino acid Arg. These hmH3034/sup3034 mutants and the separated sup3034 mutants form fully functional tailspike proteins with assembly activities indistinguishable from wild type while retaining the SDS-sensitive structural defect. From the analysis of the hmH3034 mutant and its suppressors, we propose that in the wild-type tailspike protein, the Asp residue at position 100 and the Arg residue at position 13 form an intrachain or interchain salt bridge which stabilizes the amino terminus of the tailspike protein and that the unneutralized positive charge at amino acid 13 in the hmH3034 protein is the cause of the assembly defect of this protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cysteines in CH1 underlie retention of unassembled Ig heavy chains

Conformation, structure, and oligomeric state of immunoglobulins not only control quality and functional properties of antibodies but are also critical for immunoglobulins secretion. Unassembled immunoglobulin heavy chains are retained intracellularly by delayed folding of the C(H)1 domain and irreversible interaction of BiP with this domain. Here we show that the three C(H)1 cysteines play a central role in immunoglobulin folding, assembly, and secretion. Remarkably, ablating all three C(H)1 cysteines negates retention and enables BiP cycling and non-canonical folding and assembly. This phenomenon is explained by interdependent formation of intradomain and interchain disulfides, although both bonds are dispensable for secretion. Substituting Cys-195 prevents formation not only of the intradomain disulfide, but also of the interchain disulfide bond with light chain, BiP displacement, and secretion. Mutating the light chain-interacting Cys-128 hinders disulfide bonding of intradomain cysteines, allowing their opportunistic bonding with light chain, without hampering secretion. We propose that the role of C(H)1 cysteines in immunoglobulin assembly and secretion is not simply to engage in disulfide bridges, but to direct proper folding and interact with the retention machinery.     

Structural characterization of PTX3 disulfide bond network and its multimeric status in cumulus matrix organization

PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.     

Pressure treatment of tailspike aggregates rapidly produces on-pathway folding intermediates

Protein folding and aggregation are in direct competition in living systems, yet measuring the two pathways simultaneously has rarely been accomplished. In order to identify the mechanism of high-pressure dissociation of aggregates, we compared the simultaneous on- and off-pathway behavior following dilution of freshly denatured P22 tailspike protein. Tailspike assembly at 100 microg/mL was monitored at four temperatures using a combination of size-exclusion chromatography and native polyacrylamide gel electrophoresis (PAGE) and folding and aggregation rates and yields were determined. As temperature increased, the yield of native trimeric tailspike decreased from 26.1 +/- 1.3 microg/mL at 20 degrees C to 0 microg/mL at 37 degrees C. Pressure treatment dissociated 60% of the trapped aggregates created at 37 degrees C and yielded 19.8 +/- 1.1 microg/mL of native trimer following depressurization and incubation at 20 degrees C. The rate of refolding of "freshly denatured" tailspike was compared to that following pressure treatment. The trimer formation rate increased by a factor of roughly five, and the aggregate rate decreased by a factor of three, following pressure treatment. Circular dichroism and high-pressure intrinsic tryptophan fluorescence measurements support the model that a structured intermediate is formed in a rapid manner under high pressure from a pressure-sensitive aggregate population.     

Role of a disulfide bond in the thermal stability of the LamB protein trimer in Escherichia coli outer membrane

In order to understand the unusual heat resistance of LamB protein (the outer membrane component of the maltose transport system in Escherichia coli and its receptor for bacteriophage lambda), we investigated the role of its 2 cysteinyl residues. Our studies show that Cys22 and Cys38 form an intrasubunit disulfide bond which contributes to the heat stability of the LamB protein trimer. Physical evidence for the disulfide was obtained by using site-directed mutagenesis to convert Asn36 to Met, which allowed cyanogen bromide cleavage between the 2 cysteines. Upon reduction one of the N36M fragments migrated as two pieces, resolved by two-dimensional polyacrylamide gel electrophoresis. Other mutagenized LamB proteins, in which 1 or both Cys residues were converted to Ser, exhibited a sharp loss of thermal stability. In contrast to wild-type LamB protein trimer, which does not dissociate to monomers even after 60 min at 100 degrees C, only 10-15% of the mutant LamB proteins remain trimeric after boiling 10 min. The disulfide bond in LamB protein is not required for its transport function, since both mutagenized LamB protein and N-ethylmaleimide-labeled LamB protein exhibit normal uptake of sugars in proteoliposomes. Finally, the disulfide bond must not be between subunits of the LamB trimer since reversible dissociation of trimer is achieved by low pH or denaturants in the absence of reducing agent.     

Disulfide bond formation during the folding of influenza virus hemagglutinin

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.     

Collagenous sequence governs the trimeric assembly of collagen XII

A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange between intra- and interchain bonding is observed, suggesting that the triple-helix forms first and that in favorable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of interchain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COL1 domain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the presence of five additional C-terminal triplets in COL1 allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence of a triple-helix is essential for the assembly of collagen XII.     

Properties of monoclonal antibodies selected for probing the conformation of wild type and mutant forms of the P22 tailspike endorhamnosidase

Eleven species of monoclonal antibodies directed against the trimeric P22 tailspike endorhamnosidase have been selected and characterized. Seven of these antibodies recognize the native tailspike, both isolated and assembled onto the virion, and prevent phage infection. Four antibodies react with denatured forms of the tailspike as well as with the plastic absorbed tailspike. Three of these latter prevent the tailspike from assembling onto the phage head. The antibodies have been tested against tailspike proteins carrying single amino acid substitutions at 15 different sites on the protein. Two of these mutations interfere with binding by a set of the monoclonals, indicating that they disrupt the epitopes for these antibodies. Since amino acid replacements corresponding to the temperature-sensitive folding mutations do not change the conformation of the native protein, these mutant proteins may be particularly useful for mapping epitopes. Amber fragments of the tailspike chain are recognized predominantly by the anti-denatured antibodies suggesting either that they are conformationally closer to folding intermediates than to the native tailspike or that the epitopes recognized by anti-native antibodies are carried by the C-terminal end of the native protein. Immunochemical detection by an anti-denatured antibody, after sucrose gradient sedimentation of a large 55-kDa amber fragment, indicates a monomeric rather than a trimeric state. This suggests that the missing C-terminal region is important for the trimerization reaction. Such N-terminal amber fragments may be useful models for studying with the monoclonal antibodies the nascent chain emerging from the ribosome.     

Role of disulfide bonds in folding and secretion of human lysozyme in Saccharomyces cerevisiae

We examined folding and secretion of human lysozyme using four mutants each lacking two cysteines expressed in a yeast secretion system. Our results have revealed that the formation of the disulfide bond Cys6/Cys128 in human lysozyme is a prerequisite for correct folding in vivo in yeast. Substitution of Ala for Cys77 and Cys95 gave eight-fold greater secretion of a molecule with almost the same specific activity as that of the native enzyme. Substitutions of the other cysteines gave molecules that were secreted at a lower rate and had lower specific activities than the native enzyme. These are the first findings that the individual disulfide bonds of human lysozyme have different functions in folding and secretion in vivo.