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PubMed1980

General specificity of cytoplasmic thioltransferase (thiol:disulfide oxidoreductase) from rat liver for thiol and disulfide substrates.

The substrate specificity of thioltransferase (thiol:disulfide oxidoreductase) purified from rat liver cytosol (Axelsson, K., Eriksson, S. and Mannervik, B. (1978) Biochemistry 17, 2978-2984) has been investigated. Various low-molecular-weight thiols were found to be substrates in the scission of sulfur-sulfur bonds in cystamine and glutathione disulfide as well as in mixed disulfides of proteins and glutathione. Protein sulfhydryl groups could also serve as thiol substrates in enzyme-catalyzed thiol-disulfide interchange. Thiol-transferase also catalyzed the reduction of sulfur-sulfur bonds in disulfide-containing proteins by glutathione provided that the proteins had been denatured by proteolysis or urea treatment. It is proposed that thioltransferase may have a role in the intracellular protein degradation by cleavage of disulfide bonds. The finding that thioltransferase catalyzes the reversible formation of mixed disulfides of glutathione and the bulk of proteins in rat liver cytosol is relevant for the catalysis of changes in the 'glutathione status' in the cell. In conclusion, the results of the present investigation show that cytoplasmic thiol-transferase has a very broad substrate specificity, which implies that the enzyme may participate in a multitude of thiol-disulfide interchange reactions.

Animals↗
PubMed2005

Domain architecture of protein-disulfide isomerase facilitates its dual role as an oxidase and an isomerase in Ero1p-mediated disulfide formation.

Native disulfide bond formation in eukaryotes is dependent on protein-disulfide isomerase (PDI) and its homologs, which contain varying combinations of catalytically active and inactive thioredoxin domains. However, the specific contribution of PDI to the formation of new disulfides versus reduction/rearrangement of non-native disulfides is poorly understood. We analyzed the role of individual PDI domains in disulfide bond formation in a reaction driven by their natural oxidant, Ero1p. We found that Ero1p oxidizes the isolated PDI catalytic thioredoxin domains, A and A' at the same rate. In contrast, we found that in the context of full-length PDI, there is an asymmetry in the rate of oxidation of the two active sites. This asymmetry is the result of a dual effect: an enhanced rate of oxidation of the second catalytic (A') domain and the substrate-mediated inhibition of oxidation of the first catalytic (A) domain. The specific order of thioredoxin domains in PDI is important in establishing the asymmetry in the rate of oxidation of the two active sites thus allowing A and A', two thioredoxin domains that are similar in sequence and structure, to serve opposing functional roles as a disulfide isomerase and disulfide oxidase, respectively. These findings reveal how native disulfide folding is accomplished in the endoplasmic reticulum and provide a context for understanding the proliferation of PDI homologs with combinatorial arrangements of thioredoxin domains.

Binding Sites↗
PubMed1987

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation.

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.

Animals↗
PubMed2000

Disulfide bond structure of the atrial natriuretic peptide receptor extracellular domain: conserved disulfide bonds among guanylate cyclase-coupled receptors.

The disulfide bond structure of the extracellular domain of rat atrial natriuretic peptide (ANP) receptor (NPR-ECD) has been determined by mass spectrometry (MS) and Edman sequencing. Recombinant NPR-ECD expressed in COS-1 cells and purified from the culture medium binds ANP with as high affinity as the natural ANP receptor. Reaction with iodoacetic acid yielded no S-carboxymethylcysteine, indicating that all six Cys residues in NPR-ECD are involved in disulfide bonds. Electrospray ionization MS of NPR-ECD deglycosylated by peptide-N-glycosidase F gave a molecular mass of 48377.5+/-1.6 Da, which was consistent with the presence of three disulfide bonds. Liquid chromatography MS analysis of a lysylendopeptidase digest yielded three cystine-containing fragments with disulfide bonds Cys(60)-Cys(86), Cys(164)-Cys(213) and Cys(423)-Cys(432) based on their observed masses. These bonds were confirmed by Edman sequencing of each of the three fragments. No evidence for an inter-molecular disulfide bond was found. The six Cys residues in NPR-ECD, forming a 1-2, 3-4, 5-6 disulfide pairing pattern, are strictly conserved among A-type natriuretic peptide receptors and are similar in B-type receptors. We found that in other families of guanylate cyclase-coupled receptors, the Cys residues involved in 1-2 and 5-6 disulfide pairs are conserved in nearly all, suggesting an important contribution of these disulfide bonds to the receptor's structure and function.

Amino Acid Sequence↗
PubMed2002

Reductive unfolding and oxidative refolding of a Bowman-Birk inhibitor from horsegram seeds (Dolichos biflorus): evidence for "hyperreactive" disulfide bonds and rate-limiting nature of disulfide isomerization in folding.

Horsegram protease inhibitor belongs to the Bowman-Birk class (BBIs) of low molecular weight (8-10 kDa), disulfide-rich, "dual" inhibitors, which can bind and inhibit trypsin and chymotrypsin either independently or simultaneously. They have seven conserved disulfide bonds. Horsegram BBI exhibits remarkable stability against denaturants like urea, guanidine hydrochloride (GdmCl) and heat, which can be attributed to these conserved disulfide bonds. On reductive denaturation, horsegram BBI follows the "two-state" mode of unfolding where all the disulfide bonds are reduced simultaneously resulting in the fully reduced protein without any accumulation of partially reduced intermediates. Reduction with dithiothreitol (DTT) followed apparent first-order kinetics and the rate constants (k(r)) indicated that the disulfide bonds were "hyperreactive" in nature. Oxidative refolding of the fully reduced and denatured inhibitor was possible at very low protein concentration in the presence of "redox" combination of reduced and oxidized glutathiones. Simultaneous recovery of trypsin and chymotryptic inhibitory activities indicated the concomitant folding of both the inhibitory subdomains. Folding efficiency decreased in the absence of the glutathiones and in the presence of denaturants (6 M urea and 4 M GdmCl), indicating the importance of disulfide shuffling and the formation of noncovalent interactions and secondary structural elements, respectively, for folding efficiency. Folding rate was significantly improved in the presence of protein disulfide isomerase (PDI). A 3-fold enhancement of rate was observed in the presence of PDI at molar ratio of 1:20 (PDI/inhibitor), indicating that disulfide bond formation and isomerization to be rate limiting in folding. Peptide prolyl cis-trans isomerase (PPI) did not affect rate at low concentrations, but at molar ratios of 1:1.5 (PPI/inhibitor), there was 1.4-fold enhancement of the folding rate, indicating that the prolyl imidic bond isomerizations may be slowing down the folding reaction but were not rate limiting.

Amino Acid Sequence↗
PubMed2001

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its beta-subunit: possible involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) in receptor binding of the hormone.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGbeta in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGbeta were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [125I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9-57) was found to be approximately 4-fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23-72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) of the beta-subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.

Amino Acid Sequence↗
PubMed1998

Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme.

The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily.

Bacterial Proteins↗
PubMed1989

Determination of penicillamine, penicillamine disulfide and penicillamine-glutathione mixed disulfide by high-performance liquid chromatography with electrochemical detection.

Methodology is described for the simultaneous determination of D-penicillamine, penicillamine disulfide and the penicillamine-glutathione mixed disulfide, as well as glutathione and glutathione disulfide, in human plasma, erythrocytes and urine. The various thiols and disulfides are separated by reversed-phase ion-pairing liquid chromatography with detection by an electrochemical detector with dual gold/mercury amalgam electrodes in series. The thiols are detected at the downstream electrode; the disulfides are reduced at the upstream electrode and then detected as the thiols at the downstream electrode. Detection limits (at a signal-to-noise ratio of 2.0) are in the picomole range for 20 microliters of injected solution for all compounds except penicillamine disulfide, which has a detection limit of 600 pmol in 20 microliters. A convenient method is described for preparation of the penicillamine-glutathione mixed disulfide by thiol/disulfide exchange with standardization of the solution by 1H NMR spectroscopy.

Chromatography, High Pressure Liquid↗
PubMed1999

Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum.

Native protein disulfide bond formation in the endoplasmic reticulum (ER) requires protein disulfide isomerase (PDI) and Ero1p. Here we show that oxidizing equivalents flow from Ero1p to substrate proteins via PDI. PDI is predominantly oxidized in wild-type cells but is reduced in an ero1-1 mutant. Direct dithiol-disulfide exchange between PDI and Ero1p is indicated by the capture of PDI-Ero1p mixed disulfides. Mixed disulfides can also be detected between PDI and the ER precursor of carboxypeptidase Y (CPY). Further, PDI1 is required for the net formation of disulfide bonds in newly synthesized CPY, indicating that PDI functions as an oxidase in vivo. Together, these results define a pathway for protein disulfide bond formation in the ER. The PDI homolog Mpd2p is also oxidized by Ero1p.

Carboxypeptidases↗
PubMed1995

Refolding by disulfide isomerization: the mixed disulfide between ribonuclease T1 and glutathione as a model refolding substrate.

Protein folding, associated with isomerization of disulfide bonds, was studied using the mixed disulfide between glutathione and reduced ribonuclease T1 (GS-RNase T1) as a stable soluble and homogeneous starting material; conditions were selected to model those within the lumen of the endoplasmic reticulum where native disulfide bonds are formed in protein biosynthesis. Folding was initiated by addition of free glutathione (GSH +/- GSSG) to promote thiol-disulfide interchange and was monitored by intrinsic protein fluorescence, appearance of native ribonuclease activity, HPLC, and nonreducing SDS-PAGE. All the analyses indicated that native RNase T1 was recovered in high yield in a variety of redox conditions. Appearance of native activity followed first-order kinetics; kinetic analysis of the intrinsic fluorescence changes indicated an additional rapid process in some conditions, interpreted as the formation of a nonnative intermediate state. Analysis by HPLC and SDS-PAGE also indicated the formation of transient intermediates. In 1.5 M NaCl, GS-RNase T1 adopts a compact native-like conformation; refolding by thiol-disulfide interchange in these conditions was accelerated approximately 2-fold. Refolding of GS-RNase T1 was catalyzed by protein disulfide isomerase (PDI); substoichiometric quantities of PDI accelerated refolding several-fold. GS-RNase T1 refolding was inhibited by BiP; refolding was completely blocked in presence of a 5-fold molar excess of BiP, and the yield of refolding was substantially reduced by equimolar concentrations of BiP; the refolding was then restored by the addition of ATP. GS-RNase T1 is a convenient model substrate for studying protein folding linked to native disulfide formation in conditions comparable to those within the lumen of the endoplasmic reticulum.

Animals↗
PubMed2002

Role of disulfide bonds in folding and activity of leiurotoxin I: just two disulfides suffice.

The aim of this study is to investigate the contribution of each disulfide bond in the folding and function of leiurotoxin I, a short scorpion toxin that blocks small conductance K(+) channels. The structure of leiurotoxin I contains a motif conserved in all scorpion toxins, formed by a helix and a double-stranded beta-sheet and stabilized by three disulfide bridges. We synthesized three analogues, each presenting two alpha-aminobutyric acid (Abu) moieties replacing two bridged cysteine residues: LeTx1 ([Abu 3,21] Leiurotoxin I), LeTx2 ([Abu 8,26] Leiurotoxin I), and LeTx3 ([Abu 12,28] Leiurotoxin I). All three analogues fold into a major product containing two native disulfide bonds, while LeTx3 forms an additional isomer, containing non-native disulfides. In denaturing conditions, analogues LeTx2 and LeTx3 yield non-native isomers, while LeTx1 only forms the isomer with native disulfides. All isomers with native disulfides contain nativelike alpha-helical conformations and bind to synaptosomal membranes with affinities within a log of that shown by the native toxin. By contrast, the non-native LeTx3A analogue exhibits a disordered conformation and a decreased biological potency. Our results indicate that the "CxxxC, CxC" cysteine spacing, conserved in all scorpion toxins and preserved in LeTx1, may play an active role in folding, and that only two native disulfide bonds in leiurotoxin I are sufficient to preserve a nativelike and active conformation. Thus, in the scorpion toxin scaffold, modifications of conserved and interior cysteine residues may permit modulation of function, without significantly affecting folding efficiency and structure.

Amino Acid Sequence↗
PubMed2002

Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry.

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors. Concentrations of inhibitors that prevent this reduction and inhibit the cleavage of surface-bound disulfide conjugate prevent infection at the level of HIV-1 entry. The entry of HIV-1 strains differing in their coreceptor specificities is similarly inhibited, and so is the reduction of gp120 bound to CD4 of coreceptor-negative cells. PDI inhibitors also prevent HIV envelope-mediated cell-cell fusion but have no effect on the entry of HIV-1 pseudo-typed with murine leukemia virus envelope. Importantly, PDI coprecipitates with both soluble and cellular CD4. We propose that a PDI.CD4 association at the cell surface enables PDI to reach CD4-bound virus and to reduce disulfide bonds present in the domain of gp120 that binds to CD4. Conformational changes resulting from the opening of gp120-disulfide loops may drive the processes of virus-cell and cell-cell fusion. The biochemical events described identify new potential targets for anti-HIV agents.

Animals↗
PubMed2002

Reduction-reoxidation cycles contribute to catalysis of disulfide isomerization by protein-disulfide isomerase.

Protein-disulfide isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding. This process can be error-prone in its early stages, and any incorrect disulfides that form must be rearranged to their native configuration. When the second cysteine (CGHC) in the PDI active site is mutated to Ser, the isomerase activity drops by 7-8-fold, and a covalent intermediate with the substrate accumulates. This led to the proposal that the second active site cysteine provides an escape mechanism, preventing PDI from becoming trapped with substrates that isomerize slowly (Walker, K. W., and Gilbert, H. F. (1997) J. Biol. Chem. 272, 8845-8848). Escape also reduces the substrate, and if it is invoked frequently, disulfide isomerization will involve cycles of reduction and reoxidation in preference to intramolecular isomerization of the PDI-bound substrate. Using a gel-shift assay that adds a polyethylene glycol-conjugated maleimide of 5 kDa for each sulfhydryl group, we find that PDI reduction and oxidation are kinetically competent and essential for isomerization. Oxidants inhibit isomerization and oxidize PDI when a redox buffer is not present to maintain the PDI redox state. Reductants also inhibit isomerization as they deplete oxidized PDI. These rapid cycles of PDI oxidation and reduction suggest that PDI catalyzes isomerization by trial and error, reducing disulfides and oxidizing them in a different configuration. Disulfide reduction-reoxidation may set up critical folding intermediates for intramolecular isomerization, or it may serve as the only isomerization mechanism. In the absence of a redox buffer, these steady-state reduction-oxidation cycles can balance the redox state of PDI and support effective catalysis of disulfide isomerization.

Binding Sites↗
PubMed2006

Rapid sequencing and disulfide mapping of peptides containing disulfide bonds by using 1,5-diaminonaphthalene as a reductive matrix.

MS/MS is indispensable for the amino acid sequencing of peptides. However, its use is limited for peptides containing disulfide bonds. We have applied the reducing properties of 1,5-diaminonaphthalene (1,5-DAN) as a MALDI matrix to amino acid sequencing and disulfide bond mapping of human urotensin II possessing one disulfide bond, and human guanylin possessing two disulfide bonds. 1,5-DAN was used in the same manner as the usual MALDI matrices without any pre-treatment of the peptide, and MS/MS was performed using a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer (MALDI QIT TOFMS). The results demonstrated that MS/MS of the molecular ions reduced by 1,5-DAN provided a series of significant b-/y-product ions. All 11 amino acid residues of urotensin II were identified using 1,5-DAN, while only 5 out of 11 residues were identified using 2,5-dihydroxybenzoic acid (DHB); similarly 11 out of 15 amino acid residues of guanylin were identified using 1,5-DAN, while only three were identified using DHB. In addition, comparison of the theoretical and measured values of the mass differences between corresponding MS/MS product ions using 1,5-DAN and DHB narrowed down the possible disulfide bond arrangement candidates. Consequently, 1,5-DAN as a reductive matrix facilitates rapid amino acid sequencing and disulfide mapping for peptides containing disulfide bonds.

2-Naphthylamine↗
PubMed2004

Algorithm-assisted elucidation of disulfide structure: application of the negative signature mass algorithm to mass-mapping the disulfide structure of the 12-cysteine transforming growth factor beta type II receptor extracellular domain.

The power of an algorithm-driven method for interpreting disulfide mass-mapping data is demonstrated in the context of determining the disulfide structure of the extracellular domain of the transforming growth factor beta type II receptor, a 14-kDa cystinyl protein containing 12 cysteines in the form of six disulfide bonds. The disulfide mass-mapping methodology is based on partial reduction and cyanylation-induced cleavage of the cystinyl protein. Because the multiplicity of possible disulfide structures that must be considered grows rapidly with the number of cysteines, as does the difficulty in physically isolating each of the partially reduced and cyanylated isoforms of the analyte, manual data interpretation for disulfide mapping a cystinyl protein containing more than eight cysteines becomes unmanageable. Recently, we introduced the concept of a "negative signature mass algorithm" (NSMA) to determine the disulfide structure of a cystinyl protein by processing an input of its amino acid sequence and mass spectral data from analysis of its associated cyanylation-induced cleavage products. Here, we present experimental results to validate the NSMA concept. A key advantage of the NSMA, in addition to convenience and automation, is its capacity to interpret mass spectra from mixtures of cyanylation-induced cleavage fragments without separating the partially reduced isoforms of the cystinyl protein and without knowledge of the extent of partial reduction.

Algorithms↗
PubMed2006

Disulfide isomerization and thiol-disulfide exchange of long neurotoxins from the venom of Ophiophagus hannah.

Selective reduction on the Cys28-Cys32 disulfide of Ophiophagus hannah neurotoxins, Oh-4 and Oh-5, revealed that isomerization of this disulfide linkage caused the two toxins to have distinct conformation and different retention time on a reversed-phase column. The Cys28-Cys32 disulfide of Oh-4 and Oh-5 was prone to form mixed disulfides with glutathione following pseudo-first-order kinetics. In addition to glutathionylated proteins, Oh-4 could be promoted to convert into Oh-5 by thiol compounds. Isomerization of Oh-5 into Oh-4 was not observed in the presence of thiol compounds. Dethiolation of glutathionylated proteins produced Oh-4 and Oh-5. Oxidation of the partially reduced toxin with reduced Cys28 and Cys32 was exclusively converted into Oh-5 regardless of the absence or presence of GSH/GSSG. Acrylamide quenching studies revealed difference in degree of exposure of the single Trp27 between Oh-4 and Oh-5. Synthesized peptides with substitution of Trp27 or Phe31 with Gly abolished entirely the formation of disulfide-linked dimeric product noted with the peptide of wild-type sequence. These results suggest that disulfide formation and isomerization of Cys28-Cys32 could be regulated by thiolation, and that the bulky aromatic residues Trp27 and Phe31 facilitate favorably the occurrence of disulfide isomerization of Cys28-Cys32.

Amino Acid Sequence↗
PubMed1997

Reduction of protein disulfide bonds in an oxidizing environment. The disulfide bridge of cholera toxin A-subunit is reduced in the endoplasmic reticulum.

Following retrograde transport to the endoplasmic reticulum (ER) the A-subunit of cholera toxin (CTX-A) is partially cleaved into CTX-A1 and CTX-A2 by reduction of a disulfide bridge [Majoul et al. (1996) J. Cell Biol. 133, 777-789], although the redox state in the ER favors disulfide formation. We show here that the disulfide bridge of CTX-A is cleaved in vitro already at GSH/GSSG ratios between 1 and 3. Protein disulfide isomerase (PDI) exerts only a minor accelerating effect. Various mixed disulfide intermediates (CTX-A1-S-S-CTX-A1; PDI-S-S-A2; PDI-S-S-A1) appear during CTX-A reduction. These results indicate that in the ER protein disulfide formation and protein disulfide reduction can take place simultaneously.

Animals↗
PubMed2001

Folding of a disulfide-bonded protein species with free thiol(s): competition between conformational folding and disulfide reshuffling in an intermediate of bovine pancreatic ribonuclease A.

The conformational folding of the nativelike intermediate des-[40-95] on the major oxidative folding pathway of bovine pancreatic ribonuclease A (RNase A) has been examined at various pHs and temperatures in the absence of a redox reagent. Des-[40-95] has three of the four disulfide bonds of native RNase A and lacks the bond between Cys40 and Cys95. This three-disulfide species was unfolded at low pH to inhibit any disulfide reshuffling and was refolded at higher pH, allowing both conformational folding and disulfide-reshuffling reactions to take place. As a result of this competition, 15-85% of des-[40-95], depending on the experimental conditions, undergoes intramolecular disulfide-reshuffling reactions. That portion of the des-[40-95] population which has native isomers of essential proline residues appears to fold faster than the disulfide reaction can occur. However, when the folding is retarded, conceivably by the presence of non-native isomers of essential proline residues, des-[40-95] may reshuffle before completing the conformational folding process. These results enable us to distinguish among current models for the critical structure-forming step in oxidative folding and reveal a new model for coupling proline isomerization to disulfide-bond formation. These experiments also demonstrate that the reshuffling-folding competition assay is a useful tool for detecting structured populations in conformational folding intermediates.

Animals↗