Combinations of protein-disulfide isomerase domains show that there is little correlation between isomerase activity and wild-type growth.

Protein-disulfide isomerase (PDI) has five domains: a, b, b', a' and c, all of which except c have a thioredoxin fold. A single catalytic domain (a or a') is effective in catalyzing oxidation of a reduced protein but not isomerization of disulfides (Darby, N. J., and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). To examine the structural basis for this oxidase and isomerase activity of PDI, shuffled domain mutants were generated using a method that should be generally applicable to multidomain proteins. Domains a and a' along with constructs ab, aa', aba', ab'a' display low disulfide isomerase activity, but all show significant reactivity with mammalian thioredoxin reductase, suggesting that the structure is not seriously compromised. The only domain order that retains significant isomerase activity has the b' domain coupled to the N terminus of the a' domain. This b'a'c has 38% of the isomerase activity of wild-type PDI, equivalent to the activity of full-length PDI with one of the active sites inactivated by mutation (Walker, K. W., Lyles, M. M., and Gilbert, H. F. (1996) Biochemistry 35, 1972-1980). Individual a and a' domains, despite their very low isomerase activities in vitro, support wild-type growth of a pdi1Delta Saccharomyces cerevisiae strain yeast. Thus, most of the PDI structure is dispensable for its essential function in yeast, and high-level isomerase activity appears not required for viability or rapid growth.

Discrimination between native and non-native disulfides by protein-disulfide isomerase.

The folding assistant and chaperone protein-disulfide isomerase (PDI) catalyzes disulfide formation, reduction, and isomerization of misfolded proteins. PDI substrates are not restricted to misfolded proteins; PDI catalyzes the dithiothreitol (DTT)-dependent reduction of native ribonuclease A, microbial ribonuclease, and pancreatic trypsin inhibitor, suggesting that an ongoing surveillance by PDI can test even native disulfides for their ability to rearrange. The mechanism of reduction is consistent with an equilibrium unfolding of the substrate, attack by the nucleophilic cysteine of PDI followed by direct attack of DTT on a covalent intermediate between PDI and the substrate. For native proteins, the rate constants for PDI-catalyzed reduction correlate very well with the rate constants for uncatalyzed reduction by DTT. However, the rate is weakly correlated with disulfide stability, surface exposure, or local disorder in the crystal. Compared with native proteins, scrambled ribonuclease is a much better substrate for PDI than predicted from its reactivity with DTT; however, partially reduced bovine pancreatic trypsin inhibitor (des(14-38)) is not. An extensively unfolded polypeptide may be required by PDI to distinguish native from non-native disulfides.

Hormone binding by protein disulfide isomerase, a high capacity hormone reservoir of the endoplasmic reticulum.

Protein disulfide isomerase (PDI) is a folding assistant of the eukaryotic endoplasmic reticulum, but it also binds the hormones, estradiol, and 3,3',5-triiodo-l-thyronine (T(3)). Hormone binding could be at discrete hormone binding sites, or it could be a nonphysiological consequence of binding site(s) that are involved in the interaction PDI with its peptide and protein substrates. Equilibrium dialysis, fluorescent hydrophobic probe binding (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS)), competition binding, and enzyme activity assays reveal that the hormone binding sites are distinct from the peptide/protein binding sites. PDI has one estradiol binding site with modest affinity (2.1 +/- 0.5 microm). There are two binding sites with comparable affinity for T(3) (4.3 +/- 1.4 microm). One of these overlaps the estradiol site, whereas the other binds the hydrophobic probe, bis-ANS. Neither estradiol nor T(3) inhibit the catalytic or chaperone activity of PDI. Although the affinity of PDI for the hormones estradiol and T(3) is modest, the high local concentration of PDI in the endoplasmic reticulum (>200 microm) would drive hormone binding and result in the association of a substantial fraction (>90%) of the hormones in the cell with PDI. High capacity, low affinity hormone sites may function to buffer hormone concentration in the cell and allow tight, specific binding to the true receptor while preserving a reasonable number of hormone molecules in the very small volume of the cellular environment.

A secondary drug resistance mutation of TEM-1 beta-lactamase that suppresses misfolding and aggregation.

In Gram-negative bacteria, TEM-1 beta-lactamase provides the major mechanism of plasmid-mediated beta-lactam resistance. Natural variants of TEM-1 with increased antibiotic resistance have appeared in response to the use of extended-spectrum beta-lactam antibiotics (e.g., ceftazidime) and beta-lactamase inhibitors (e.g., clavulanic acid). Some of the variant enzymes are more efficient at catalyzing beta-lactam hydrolysis, whereas others are more resistant to inhibitors. M182T is a substitution observed in both types of variant TEM-1 beta-lactamases. This mutation is found only in combination with other amino acid substitutions, suggesting that it may correct defects introduced by other mutations that alter the specificity. An engineered core mutation, L76N, which diminishes the periplasmic beta-lactamase activity by 100-fold, was used as a model to understand the mechanism of suppression of the M182T mutation. Biochemical studies of the L76N enzyme alone and in combination with the M182T mutation indicate that the M182T substitution acts at the level of folding but does not affect the thermodynamic stability of TEM-1 beta-lactamase. Thus, the M182T substitution is an example of a naturally occurring mutation that has evolved to alter the folding pathway of a protein and confer a selective advantage during the evolution of drug resistance.

Mechanism of the antichaperone activity of protein disulfide isomerase: facilitated assembly of large, insoluble aggregates of denatured lysozyme and PDI.

Protein disulfide isomerase (PDI), a folding catalyst and chaperone can, under certain conditions, facilitate the misfolding and aggregation of its substrates. This behavior, termed antichaperone activity [Puig, A., and Gilbert, H. F., (1994) J. Biol. Chem. 269, 25889] may provide a common mechanism for aggregate formation in the cell, both as a normal consequence of cell function or as a consequence of disease. When diluted from the denaturant, reduced, denatured lysozyme (10-50 microM) remains soluble, although it does aggregate to form an ensemble of species with an average sedimentation coefficient of 23 +/- 5 S (approximately 600 +/- 100 kDa). When low concentrations of PDI (1-5 microM) are present, the majority (80 +/- 8%) of lysozyme molecules precipitate in large, insoluble aggregates, together with 87 +/- 12% of the PDI. PDI-facilitated aggregation occurs even when disulfide formation is precluded by the presence of dithiothreitol (10 mM). Maximal lysozyme-PDI precipitation occurs at a constant lysozyme/PDI ratio of 10:1 over a range of lysozyme concentrations (10-50 microM). Concomitant resolubilization of PDI and lysozyme from these aggregates by increasing concentrations of urea suggests that PDI is an integral component of the mixed aggregate. PDI induces lysozyme aggregation by noncovalently cross-linking 23 S lysozyme species to form aggregates that become so large (approximately 38,000 S) that they are cleared from the analytical ultracentrifuge even at low speed (1500 rpm). The rate of insoluble aggregate formation increases with increasing PDI concentration (although a threshold PDI concentration is observed). However, increasing lysozyme concentration slows the rate of aggregation, presumably by depleting PDI from solution. A simple mechanism is proposed that accounts for these unusual aggregation kinetics as well as the switch between antichaperone and chaperone behavior observed at higher concentrations of PDI.

Facilitating the formation of disulfide bonds in the Escherichia coli periplasm via coexpression of yeast protein disulfide isomerase.

Sacchromyces cerevisiae protein disulfide isomerase (yPDI) was expressed in the E. coli periplasm by using plasmids encoding the OmpA-yPDI-(His)(6) fusion gene under the control of the araBAD, trc, or T7 promoter. The expression levels of yeast PDI under these promoters were compared. Our results showed that yeast PDI expressed into the periplasm could catalyze the formation of disulfide bonds in alkaline phosphatase, restoring the phoA(+) phenotype in dsbA(-) mutants. The yeast PDI was purified from the Escherichia coli periplasm and shown to exhibit catalytic properties comparable to those of the rat enzyme with reduced RNase as substrate. In vivo, coexpression of the yeast PDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) in E. coli by 2-fold, similar to the effect seen previously with the coexpression of the rat enzyme. However yeast PDI was more effective than rat PDI in facilitating the expression of active tissue plasminogen activator (tPA). These results point to differences in the substrate specificity of various PDI enzymes, at least in the context of the E. coli periplasm.

Sulfhydryl oxidase from egg white. A facile catalyst for disulfide bond formation in proteins and peptides.

Both metalloprotein and flavin-linked sulfhydryl oxidases catalyze the oxidation of thiols to disulfides with the reduction of oxygen to hydrogen peroxide. Despite earlier suggestions for a role in protein disulfide bond formation, these enzymes have received comparatively little general attention. Chicken egg white sulfhydryl oxidase utilizes an internal redox-active cystine bridge and a FAD moiety in the oxidation of a range of small molecular weight thiols such as glutathione, cysteine, and dithiothreitol. The oxidase is shown here to exhibit a high catalytic activity toward a range of reduced peptides and proteins including insulin A and B chains, lysozyme, ovalbumin, riboflavin-binding protein, and RNase. Catalytic efficiencies are up to 100-fold higher than for reduced glutathione, with typical K(m) values of about 110-330 microM/protein thiol, compared with 20 mM for glutathione. RNase activity is not significantly recovered when the cysteine residues are rapidly oxidized by sulfhydryl oxidase, but activity is efficiently restored when protein disulfide isomerase is also present. Sulfhydryl oxidase can also oxidize reduced protein disulfide isomerase directly. These data show that sulfhydryl oxidase and protein disulfide isomerase can cooperate in vitro in the generation and rearrangement of native disulfide pairings. A possible role for the oxidase in the protein secretory pathway in vivo is discussed.

In vivo and in vitro function of the Escherichia coli periplasmic cysteine oxidoreductase DsbG.

We have characterized in vivo and in vitro the recently identified DsbG from Escherichia coli. In addition to sharing sequence homology with the thiol disulfide exchange protein DsbC, DsbG likewise was shown to form a stable periplasmic dimer, and it displays an equilibrium constant with glutathione comparable with DsbA and DsbC. DsbG was found to be expressed at approximately 25% the level of DsbC. In contrast to earlier results (Andersen, C. L., Matthey-Dupraz, A., Missiakas, D., and Raina, S. (1997) Mol. Microbiol. 26, 121-132), we showed that dsbG is not essential for growth and that dsbG null mutants display no defect in folding of multiple disulfide-containing heterologous proteins. Overexpression of DsbG, however, was able to restore the ability of dsbC mutants to express heterologous multidisulfide proteins, namely bovine pancreatic trypsin inhibitor, a protein with three disulfides, and to a lesser extent, mouse urokinase (12 disulfides). As in DsbC, the putative active site thiols in DsbG are completely reduced in vivo in a dsbD-dependent fashion, as would be expected if DsbG is acting as a disulfide isomerase or reductase. However, the latter is not likely because DsbG could not catalyze insulin reduction in vitro. Overall, our results indicate that DsbG functions primarily as a periplasmic disulfide isomerase with a narrower substrate specificity than DsbC.

Scanning and escape during protein-disulfide isomerase-assisted protein folding.

During oxidative protein folding, efficient catalysis of disulfide rearrangements by protein-disulfide isomerase is found to involve an escape mechanism that prevents the enzyme from becoming trapped in covalent complexes with substrates that fail to rearrange in a timely fashion. Protein-disulfide isomerase mutants with only a single active-site cysteine catalyze slow disulfide rearrangements and become trapped in a covalent complex with substrate. Escape is mediated by the second, more carboxyl-terminal cysteine at the active site. A glutathione redox buffer increases the kcat for single-cysteine mutants by 20-40-fold, but the presence of the second cysteine at the active site in the wild-type enzyme increases the kcat by over 200-fold. A model is developed in which kinetic scanning for disulfides of increasing reactivity is timed against an intramolecular clock provided by the second cysteine at the active site. This provides an alternative, more efficient mechanism for rearrangement involving the reduction and reoxidation of substrate disulfides.

A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities.

A catalyst of disulfide formation and isomerization during protein folding, protein-disulfide isomerase (PDI) has two catalytic sites housed in two domains homologous to thioredoxin, one near the N terminus and the other near the C terminus. The thioredoxin domains, by themselves, can catalyze disulfide formation, but they are unable to catalyze disulfide isomerizations (Darby, N. J. and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). A 21-kDa, C-terminal fragment of PDI (amino acids 308-491), termed weePDI, comprises the C-terminal third of the molecule. The kcat for ribonuclease oxidative folding by weePDI is 0.26 +/- 0.02 min-1, 3-fold lower than the wild-type enzyme but indistinguishable from the activity of a full-length mutant of PDI in which both active site cysteines of the N-terminal thioredoxin domain have been mutated to serine. Eliminating the ability of weePDI to escape easily from covalent complexes with substrate by mutating the active site cysteine nearer the C terminus to serine has a large effect on the isomerase activity of weePDI compared with its effect on the full-length enzyme. weePDI also displays chaperone and anti-chaperone activity characteristic of the full-length molecule. As isolated, weePDI is a disulfide-linked dimer in which the single cysteine (Cys-326) outside active site cross-links two weePDI monomers. The presence of the intermolecular disulfide decreases the activity by more than 2-fold. The results imply that the functions of the core thioredoxin domains of PDI and other members of the thioredoxin superfamily might be modified quite easily by the addition of relatively small accessory domains.

Facilitated protein aggregation. Effects of calcium on the chaperone and anti-chaperone activity of protein disulfide-isomerase.

Protein disulfide-isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding in the eukaryotic endoplasmic reticulum. At high concentrations, it also serves as a chaperone and inhibits aggregation. However, at lower concentrations, PDI can display the unusual ability to facilitate aggregation, termed anti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771). Under reducing conditions (10 mM dithiothreitol) and at a low concentration (0.1-0. 3 microM) relative to the unfolded protein substrate, PDI facilitates aggregation of alcohol dehydrogenase (11 microM) that has been denatured thermally or chemically. But at higher concentrations (>0.8 microM), PDI inhibits aggregation under the same conditions. With denatured citrate synthase, PDI does not facilitate aggregation, but higher concentrations do inhibit aggregation. Anti-chaperone behavior is associated with the appearance of both PDI and substrate proteins in insoluble complexes, while chaperone behavior results in the formation of large (>500 kDa) but soluble complexes that contain both proteins. Physiological concentrations of calcium and magnesium specifically increase the apparent rate of PDI-dependent aggregation and shift the chaperone activity to higher PDI concentrations. However, calcium has no effect on the Km or Vmax for PDI-catalyzed oxidative folding, suggesting that the interactions that lead to chaperone/anti-chaperone behavior are distinct from those required for catalytic activity. To account for this unusual behavior of a folding catalyst, a model with analogy to classic immunoprecipitation is proposed; multivalent interactions between PDI and a partially aggregated protein stimulate further aggregate formation by noncovalently cross-linking smaller aggregates. However, at high ratios of PDI to substrate, cross-linking may be inhibited by saturation of the sites with PDI. The effects of PDI concentration on substrate aggregation and the modulation of the behavior by physiological levels of calcium may have implications for the involvement of PDI in protein folding, aggregation, and retention in the endoplasmic reticulum.

Mycobacterium tuberculosis 16-kDa antigen (Hsp16.3) functions as an oligomeric structure in vitro to suppress thermal aggregation.

Tuberculosis continues to be a major disease threatening millions of lives worldwide. Several antigens of Mycobacterium tuberculosis, identified by monoclonal antibodies, have been cloned and are being exploited in the development of improved vaccines and diagnostic reagents. We have expressed and purified the 16-kDa antigen, an immunodominant antigen with serodiagnostic value, which has been previously cloned and shown to share low sequence homology with the alpha-crystallin-related small heat shock protein family. Sedimentation equilibrium analytical ultracentrifugation and dynamic light scattering demonstrate the formation of a specific oligomer, 149 +/- 8 kDa, consisting of approximately nine monomers. In 4 M urea, a smaller oligomer of 47 +/- 6 kDa (or trimer) is produced. Analysis by electron cryomicroscopy reveals a triangular shaped oligomeric structure arising from the presence of three subparticles or globules. Taken together, the data suggest an antigen complex structure of a trimer of trimers. This antigen, independent of ATP addition, effectively suppresses the thermal aggregation of citrate synthase at 40 degrees C, indicating that it can function as a molecular chaperone in vitro. A complex between the antigen and heat-denatured citrate synthase can be detected and isolated using high performance liquid chromatography. We propose to rename the 16-kDa antigen Hsp16.3 to be consistent with other members of the small heat shock protein family.

Catalysis of oxidative protein folding by mutants of protein disulfide isomerase with a single active-site cysteine.

Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol. Chem. 269, 30946-30952]. Each dithiol/disulfide active-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a single active-site cysteine. Kinetic analysis of these mutants show that the first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide formation or rearrangement, even at concentrations of 25 microM. The second (more C-terminal) cysteine is not essential for catalysis of RNase disulfide rearrangements, but it is essential for catalysis of RNase oxidation, even in the presence of a glutathione redox buffer. Mutant active sites with the sequence CGHS show 12%-50% of the kcat activity of wild-type active sites during the rearrangement phase of RNase refolding but < 5% activity during the oxidation phase. In addition, mutants with the sequence CGHS accumulate significant levels of a covalent PDI-RNase complex during steady-state turnover while the wild-type enzyme and mutants with the sequence SGHC do not. Since both active-site cysteines are essential for catalysis of disulfide formation, the dominant mechanism for RNase oxidation may involve direct oxidation by the active-site PDI disulfide. Although it is not essential for catalysis of RNase rearrangements, the more C-terminal cysteine does contribute 2-8-fold to the rearrangement activity. A mechanism for substrate rearrangement is suggested in which the second active-site cysteine provides PDI with a way to "escape" from covalent intermediates that do not rearrange in a timely fashion. The second active-site cysteine may normally serve the wild-type enzyme as an internal clock that limits the time allowed for intramolecular substrate rearrangements.

Oxidation of kinetically trapped thiols by protein disulfide isomerase.

The formation of a stabilized structure during oxidative protein folding can severely retard disulfide formation if the structure must be disrupted to gain access to buried cysteines. These kinetic traps can slow protein folding and disulfide bond formation to the extent that unassisted folding is too slow to be kinetically competent in the cell. Protein disulfide isomerase (PDI) facilitates the oxidation of a kinetically trapped state of RTEM-1 beta-lactamase in which two cysteines that form the single disulfide bond in the native protein are buried and approximately 500-fold less reactive than exposed cysteines. Under second-order conditions, PDI-dependent oxidation of reduced, folded beta-lactamase is 500-fold faster than GSSG-dependent oxidation. The rate difference observed between PDI and GSSG can be accounted for by the 520-fold higher kinetic reactivity of PDI as an oxidant. Noncovalent interactions between PDI (35 microM) and beta-lactamase increase the reactivity or unfolding of beta-lactamase in the steady-state by less than 3-fold. At high concentrations of PDI or alkylating agents, the reaction of beta-lactamase cysteines approaches a constant rate, limited by the spontaneous unfolding of the protein (kunfold = 0.024 +/- 0.005 min-1). PDI does not substantially increase the rate of beta-lactamase unfolding; however, once beta-lactamase spontaneously unfolds, PDI at concentrations greater than 44 +/- 4 microM, oxidizes the unfolded substrate before it can refold (kfold = 1.5 +/- 0.2 min-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in the thioredoxin sites of protein disulfide isomerase reveal functional nonequivalence of the N- and C-terminal domains.

Protein disulfide isomerase (PDI), a foldase of the endoplasmic recticulum, is a multifunctional protein that catalyzes the formation and isomerization of disulfide bonds during protein folding. The wild-type protein contains two redox active thiol/disulfide sites near the N and C terminus that are homologous to the redox center of thioredoxin. Using site-directed mutagenesis, both cysteines of each of the thioredoxin-like centers, (C35S,C38S) and (C379S,C382S) were replaced by serines. In addition, a mutant PDI was constructed with all four of the active cysteines mutated to serine (C35S,C38S,C379S,C382S). The activity of the wild-type and mutant proteins in the oxidative renaturation of reduced, denatured RNase was analyzed over a wide range of RNase concentrations, PDI concentrations, and glutathione redox buffers compositions. All mutants, including the construct with no functional thioredoxin centers, have measurable disulfide isomerase activity. Both of the thioredoxin-like sites contribute some to apparent steady-state binding (Km) and catalysis at saturating substrate concentrations (kcat); however, their contributions are not equivalent. At saturating concentrations of RNase, the mutant with an inactivated C-terminal active site (kcat = 0.72 +/- 0.06 min-1) retains near wild-type activity (kcat = 0.76 +/- 0.02 min-1), while the N-terminal mutant exhibits a significantly lower kcat (0.24 +/- 0.01 min-1). The Km for RNase is elevated for the C-terminal mutant (Km = 29 +/- 4 microM) while the N-terminal mutant (Km = 7.1 +/- 1.1 microM) exhibits a wild-type Km (6.9 +/- 0.8 microM). The larger Km for the C-terminal mutant (4.2 times wild-type) and the lower kcat of N-terminal mutant (32% of wild-type) suggest that the C-terminal region contributes more to apparent steady-state substrate binding, and the N-terminal region contributes more to catalysis at saturating concentrations of substrate. Despite their complementary roles in catalysis, the thioredoxin-like centers exhibit the same dependence on the glutathione redox buffer composition as evidenced by the equivalent K(ox) values for the wild-type (47 +/- 1 microM), N-terminal mutant (43 +/- 3 microM), and C-terminal mutant (44 +/- 1 microM). The mutant with both thioredoxin sites mutated displays a low but detectable level of disulfide-isomerase activity (0.5% of wild-type) that can be observed at high PDI concentrations. At high RNase concentrations (> or = 26 microM), wild-type PDI and all of the mutants catalyze intermolecular RNase aggregation in a nucleation growth reaction that is first order in PDI but fourth order with respect to RNase.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of redox environment on the in vitro and in vivo folding of RTEM-1 beta-lactamase and Escherichia coli alkaline phosphatase.

The oxidative folding mechanisms of two Escherichia coli periplasmic proteins, alkaline phosphatase and RTEM-1 beta-lactamase, have been examined in vitro and in vivo. In contrast to eukaryotic proteins, which require a relatively reducing environment for optimal folding rates, both alkaline phosphatase and beta-lactamase fold fastest under very oxidizing conditions. For example, bovine pancreatic ribonuclease exhibits an optimal folding rate in a redox buffer consisting of 1 mM GSH and 0.2 mM GSSG (Lyles, M. M., and Gilbert, H. F. (1991) Biochemistry 30, 613-619); however, both E. coli alkaline phosphatase and beta-lactamase exhibit optimal in vitro folding rates at low concentrations of GSH (< 0.4 mM) and very high concentrations of GSSG (4-8 mM). For both bacterial proteins, GSH inhibits oxidative folding. Under optimal redox conditions, the rate-limiting step for the in vitro oxidative folding of alkaline phosphatase depends on the concentration of the protein, consistent with a mechanism involving rapid oxidation followed by slow dimerization. With beta-lactamase, the oxidative folding mechanism involves a competition between disulfide bond formation and folding of the molecule into a catalytically active conformation that buries the 2 reduced cysteines in the core of the enzyme. The effects of including a thiol reductant in the growth medium on the in vivo folding of alkaline phosphatase and beta-lactamase are similar to the effects observed during in vitro folding of these enzymes. The levels of both oxidized proteins are decreased by GSH in the growth medium. However, addition of a disulfide oxidant to the growth medium does not positively affect the production of either enzyme. These observations are consistent with the idea that the oxidative folding mechanisms of E. coli periplasmic proteins and, by inference, proteins of the eukaryotic endoplasmic reticulum have evolved to accommodate constraints placed on the folding reaction by the folding environment. The consequences of differences between the folding mechanisms in eukaryotic and prokaryotic disulfide-containing proteins on the expression of eukaryotic proteins in the bacterial periplasm are discussed.

Protein chaperones and protein folding.

A universal strategy for obtaining maximal protein expression or refolding remains elusive; however, headway has been made toward understanding these processes in vivo. The observation of reversible protein aggregation, asymmetry in protein-chaperone complexes, redox effects on disulfide formation, and the sequential involvement of multiple chaperones and foldases may suggest new approaches. Such new approaches include immobilized catalysts and manipulation of the bacterial periplasm.