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PubMed1983

In vitro formation of disulfide-bonded fibronectin multimers.

Fibronectin purified from a plasma protein side fraction in the absence of denaturant contained 1.5 to 1.9 cryptic free sulfhydryl groups per 200- to 250-kDa subunit. Exposure of sulfhydryl groups in physiologic salt solutions required at least 1 M guanidine, and 3 M guanidine was required for optimal exposure. The sulfhydryl groups were not exposed by collagen, a fibronectin-binding collagen fragment, fibrinogen, heparin, hyaluronic acid, calcium ion, EDTA, deoxycholate, or methylamine. One- and two-dimensional gel electrophoresis indicated that a molecule of 40-60 kDa was disulfide-bonded to a minor portion of the fibronectin in whole human plasma and in preparations of purified fibronectin. In addition, traces of disulfide-bonded multimers were present in preparations of purified fibronectin. The proportion of fibronectin in disulfide-bonded multimers increased in guanidine-containing solutions. Compared to dimeric fibronectin, these multimers had limited solubility in physiologic buffers, could be readily cross-linked by Factor XIIIa, and exhibited altered tryptic susceptibility. In free sulfhydryl groups were blocked by prior alkylation with N-ethylmaleimide or iodoacetamide, fibronectin did not form disulfide-bonded multimers in guanidine-containing solutions. The patterns of altered tryptic susceptibility and cyanide cleavage suggested that multimer formation is mediated by both sulfhydryls of fibronectin. The transition from dimeric to multimeric fibronectin can serve as a model for the formation of disulfide-bonded fibronectin multimers in the extracellular matrix.

Disulfides↗
PubMed1984

Mechanism of formation of disulfide-bonded multimers of plasma fibronectin in cell layers of cultured human fibroblasts.

The free sulfhydryl groups of purified plasma fibronectin were blocked by alkylation with iodoacetamide in 3 M guanidine hydrochloride at pH 7.0 (to form fCam-fibronectin). 125I-fCam-fibronectin bound to cell layers of cultured human fibroblasts with kinetics similar to binding of 125I-labeled underivatized fibronectin. Once bound to the cell surface, 125I-fCam-fibronectin, like 125I-fibronectin, became incorporated into a detergent-insoluble extracellular matrix. Matrix-bound 125I-fCam-fibronectin, like matrix-bound 125I-fibronectin, was present as disulfide-bonded multimers as well as disulfide-bonded dimers. These results indicate that the mechanism of formation of disulfide-bonded fibronectin multimers does not involve oxidation of free sulfhydryls. Catheptic D digests of matrix-bound fibronectin were analyzed by gel electrophoresis without and with reduction. In non-reduced digests, the disulfide-rich 70-kDa amino-terminal fragment was found in a large (greater than 300 kDa) complex. Thus, fibronectin multimer formation probably occurs by disulfide exchange in the amino-terminal portion of the molecule.

Cathepsin D↗
PubMed1980

Reduction and carboxamidomethylation of the single disulfide bond of proteinase inhibitor I from potato tubers. Effects on stability, immunological properties, and inhibitory activities.

The single disulfide bond in potato Inhibitor I protomers (Mr = 8,000) was reduced and alkylated in the absence of denaturants to the carboxamidomethyl cysteine derivative. Two half-cystines per mol of inhibitor protomer were modified as determined by (a) loss of two sulfhydryl groups per reduced protomer, (b) incorporation of 2 mol of [14C]iodoacetamide per protomer, and (c) amino acid analyses. The alkylated Inhibitor I retained its oligomeric structure (Mr = 40,000) and fully retained its immunological cross reactivity with anti-Inhibitor I serum. Furthermore, the inhibitory properties of modified and unmodified Inhibitor I toward chymotrypsin were identical. The stoichiometric complex between modified Inhibitor I and chymotrypsin was stable at pH 8.0 for over 72 h. The modification of the disulfide bond introduced a lability to both heat and proteolytic enzymes. Ultraviolet difference spectra and near ultraviolet circular dichroism spectra at pH 8.0, between modified and unmodified Inhibitor I, revealed only minor absorption changes due to modification. However, in circular dichroism spectra at pH 2.0, the modified inhibitor exhibited a significant loss of absorption in the region between 270 and 280 nm that was present in the unmodified inhibitor. The cumulative results of this study indicate that the single disulfide bond in Inhibitor I, which forms a rather large disulfide loop between residues 5 and 51, is not important at neutral pH for maintaining major structural conformations which affect immunological or inhibitory activities, but the disulfide bond does impose restraints on the protein which stabilize it toward thermal denaturation and proteolysis. The susceptibility of the reduced inhibitor to plant sulfhydryl enzymes may have some importance in in vivo degradation.

Alkylation↗
PubMed1982

Intermolecular disulfide bonding of lens membrane proteins during human cataractogenesis.

Two-dimensional diagonal electrophoresis has been used to characterize intermolecular disulfide bonding of membrane proteins from lenses of cataractous and normal patients. A component of approximately 18,000 daltons, linked via intermolecular disulfide bonding, was found in membrane preparations from 10 of 17 cataracts studied. In comparison, membrane from only 1 of 12 normal lenses of approximately the same age range was found to contain intermolecular disulfide bonding of a component of similar molecular weight. Treatment of normal lens with the oxidizing agents cupric sulfate and 1,10-phenanthroline resulted in intermolecular disulfide bonding of the 18K component in a manner similar to that found in cataractous lenses. Together these results demonstrate that human cataractogenesis is many times accompanied by intermolecular disulfide bonding of a membrane component of 18K and suggest that this intermolecular bonding may be the result of the previously reported oxidative insult of the lens during human cataract formation.

Adult↗
PubMed1982

The biosynthesis of rat serum albumin. In vivo studies on the formation of the disulfide bonds.

In order to learn at what stage the disulfide bonds of albumin are formed during its biosynthesis, we perfused rat livers with iodoacetamide and then isolated the intracellular precursor, proalbumin, from organelles known to be in the pathway of albumin synthesis and secretion. The alkylated cysteines in proalbumin were determined as a measure of its thiol groups in vivo. Proalbumin of smooth microsomes was found to contain a single thiol, which is proposed to be the noncoupling cysteine occurring residue 34 in circulating albumin. Proalbumin in rough microsomes contained an average of two cysteines; the additional cysteine thiol was largely situated in the COOH-terminal region and disappeared rapidly after blocking albumin synthesis with cycloheximide. In nascent chains of proalbumin, about 45% of the cysteine + cystine was in the thiol form. From these findings we propose that disulfide bond formation begins while the nascent chain is still attached to the ribosome and proceeds in an NH2 to COOH direction. The disulfide bonding apparently is completed into the endoplasmic reticulum. Possible intermediates in the process such as mixed disulfide forms of proalbumin with glutathione or cystamine were not detected. We suggest that cysteine-34 does not participate in disulfide bonding because the NH2 terminus of proalbumin remains loosely bound to the membrane, attached by a hydrophobic segment of the chain at residues 21-27.

Animals↗
PubMed1993

In vivo control of redox potential during protein folding catalyzed by bacterial protein disulfide-isomerase (DsbA).

The formation of disulfide bonds in Escherichia coli is catalyzed by periplasmic protein disulfide-isomerase (DsbA). When the alpha-amylase/trypsin inhibitor from Ragi, a protein containing five intramolecular disulfide bridges, is secreted into the periplasm of E. coli, large amounts of misfolded inhibitor with incomplete or incorrect disulfides are accumulated. Folding of the inhibitor in the periplasm is not improved when DsbA is coexpressed and cosecreted. However, an up to 14-fold increase in correctly folded inhibitor is observed by co-expression of DsbA in conjugation with the addition of reduced glutathione to the growth medium. This peptide acts as a disulfide-shuffling reagent and can pass the outer membrane of E. coli. Since the influence of DsbA on the folding yield of the inhibitor is reduced in the presence of oxidized glutathione, the in vivo function of DsbA appears to be dependent on the ratio between oxidizing and reducing thiol equivalents in the periplasm. The high stability of thiol reagents against air oxidation during growth of E. coli allows the investigation of oxidative protein folding in vivo under controlled, thiol-dependent redox conditions.

Amino Acid Sequence↗
PubMed1994

Protein disulfide isomerase exhibits chaperone and anti-chaperone activity in the oxidative refolding of lysozyme.

Reduced, denatured lysozyme tends to aggregate at neutral pH, and competition between productive folding and aggregation substantially reduces the efficiency of refolding (Goldberg, M.E., Rudolph, R., and Jaenicke, R. (1991) Biochemistry 30, 2790-2797). Protein disulfide isomerase (PDI), a catalyst of oxidative protein folding, has a variety of effects on the yield of native lysozyme during the oxidative refolding of the reduced, denatured protein. Depending on the concentration of lysozyme, the concentration of PDI, and the order in which lysozyme and PDI are added to initiate folding, PDI can produce a substantial increase or a substantial decrease in the recovery of native lysozyme, when compared with the uncatalyzed reaction. In the presence of a glutathione redox buffer, denatured lysozyme (1-10 microM) partitions almost equally between productive folding leading to native lysozyme (50-63%) and non-productive fates including the formation of disulfide cross-linked aggregates. At the higher lysozyme concentrations examined (5-10 microM), substoichiometric concentrations of PDI (0.5-1 microM) exhibit "anti-chaperone" activity; PDI actively diverts most of the denatured lysozyme away from productive folding so that only 17 +/- 9% of the lysozyme is recovered as native enzyme. PDI's anti-chaperone activity results in extensive intermolecular disulfide crosslinking of lysozyme into large, inactive aggregates. On the other hand, if PDI is initially present at a large molar excess (5-10-fold) when denatured lysozyme is diluted to initiate folding, PDI demonstrates a chaperone-like activity that prevents aggregate formation and promotes correct folding. When PDI's chaperone activity is dominant, virtually all of the denatured lysozyme is correctly folded. The schizophrenic chaperone/anti-chaperone nature of PDI activity accounts for a number of observations on in vivo protein folding, including the necessity for maintaining a high concentration of PDI in the endoplasmic reticulum and the formation of disulfide cross-linked aggregates in the endoplasmic reticulum during the expression of disulfide-containing proteins (deSilva, A., Braakman, I., and Helenius, A. (1993) J. Cell. Biol. 120, 647-655).

Animals↗
PubMed1994

Modulation of the activity of mitochondrial aspartate aminotransferase H352C by the redox state of the engineered interdomain disulfide bond.

Molecular modeling suggested that the large and small domain of mitochondrial aspartate aminotransferase might be linked by an engineered disulfide bond that could be expected to interfere with ligand-induced and syncatalytic changes in conformation and thus to assist in the elucidation of their significance for the catalytic mechanism. His-352, which is situated in the small domain close to Cys-166 of the large domain, was replaced with a cysteine residue by oligonucleotide-directed mutagenesis. Aspartate aminotransferase H352C, that had not been exposed to reducing conditions, in part contained a disulfide bond between Cys-166 and Cys-352. Exposure to a reducing agent cleaved the crosslink completely and produced an enzyme derivative with 8% of the activity of the wild type enzyme. Cu2+-mediated autoxidation resulted in complete formation of the disulfide bond and a decrease in enzymic activity to 2%. Independently of the redox state of the disulfide bond, the H352C substitution seems to shift the equilibrium from the open toward the closed conformation of the enzyme. This change in conformation was accompanied by an increase in the binding affinity for both the amino and oxo acid substrate by one order of magnitude. Apparently, 1-2 kcal/mol of the binding energy of the substrates are no longer diverted to shift the conformational equilibrium toward the closed conformation. The kcat/Km values were unchanged or even increased in the reduced form of the mutant enzyme and only slightly decreased in its oxidized form. Both the disulfide-independent decrease in enzymic activity, as observed in reduced aspartate aminotransferase H352C and also in two other mutant enzymes (C166H/H352C and H352Q), and the redox-dependent modulation of activity indicate that unhindered domain movements are essential for full catalytic competence of aspartate aminotransferase.

Animals↗
PubMed1994

A "molten globule" of Torpedo acetylcholinesterase undergoes thiol-disulfide exchange.

Torpedo acetylcholinesterase is a disulfide-linked homodimer containing three intramolecular disulfide bonds, as well as a single free thiol on Cys-231. We report that in a "molten globule" state, produced by 1.5 M guanidine hydrochloride, this enzyme undergoes rapid intramolecular thiol-disulfide exchange, in the absence of reducing agents, resulting in the production of novel species. Most strikingly, this results in appearance of enzyme monomers. Chemical modification of the free thiol group prevents these changes. Unfolded acetylcholinesterase, namely in 5 M guanidine hydrochloride, also undergoes intramolecular thiol-disulfide exchange, including production of enzyme monomers, but at a much lower rate. Our data show that the molten globule state, in contrast to the native and unfolded states, is both compact and flexible, thus being especially amenable to thiol-disulfide exchange.

Acetylcholinesterase↗
PubMed1994

Ab initio association with beta 2-microglobulin during biosynthesis of the H-2Ld class I major histocompatibility complex heavy chain promotes proper disulfide bond formation and stable peptide binding.

In vitro translation studies indicate that the beta 2-microglobulin (beta 2-m) light chain influences the formation of intrachain disulfide bonds in class I major histocompatibility complex (MHC) molecules during their biosynthesis. We now have examined the influence of beta 2-m on class I MHC intrachain disulfide bond formation in vivo. Using beta 2-m+ and beta 2-m- derivatives of a cell line transfected with the mouse H-2Ld gene, we show that all of the H-2Ld molecules from beta 2-m+ cells have both the alpha 2 and alpha 3 intrachain disulfide bonds, whereas about 50% of the H-2Ld molecules from beta 2-m- cells have only one of these bonds. All of the free H-2Ld heavy chains from beta 2-m+ cells can undergo a peptide-induced conformational change and can bind exogenous peptide and beta 2-m stably in vitro. Only those H-2Ld molecules from beta 2-m- cells, which have both intrachain disulfide bonds, undergo a peptide- and beta 2-m-induced conformational change in vitro. These H-2Ld molecules do not bind beta 2-m and peptide stably in vitro. From these results emerges a greater understanding of the role of beta 2-m at the time of class I MHC molecule synthesis: beta 2-m promotes intrachain disulfide bond formation in the class I MHC molecule and additionally affects class I MHC structure to render it competent to form stable trimolecular complexes with peptide and beta 2-m.

Amino Acid Sequence↗
PubMed1994

Uncoupled steps of the colicin A pore formation demonstrated by disulfide bond engineering.

Four disulfide bonds were engineered into the pore-forming domain of colicin A to probe the conformational changes associated with its membrane insertion and channel formation. The soluble pore-forming domain consists of 10 alpha-helices with two outer layers (helices 1, 2, and 3-7, respectively) sandwiching a middle layer of three helices (8-10). Helices 8 and 9 form a hairpin which is completely buried and consists of hydrophobic and neutral residues only. This helical hairpin has been hypothesized to be the membrane anchor. Each double-cysteine mutant possessing an individual disulfide bond, cross-linking either helices 1 to 9 (H1/H9), 5 to 6 (H5/H6), 7 to 8 (H7/H8), or 9 to 10 (H9/H10), respectively, is unable to promote K+ efflux from sensitive Escherichia coli cells. Activity can be restored by addition of a reducing agent. In vitro studies with brominated lipid vesicles and planar lipid bilayers show that the disulfide bond which connects the helices 1 to 9 prevents colicin A membrane insertion, whereas the other disulfide bond mutants insert readily into lipid vesicles. All of the engineered bridges prevented the formation of a conducting channel in the presence of a membrane potential. This novel approach indicates that membrane insertion and channel formation are two separate steps. Moreover, the effects of the distance constraints introduced by the different disulfide bonds on colicin A activity indicate that the helical pair 1 and 2 moves away from the other helices upon membrane insertion. Helices 3-10 remain associated together. As a consequence, the results imply that the helical hairpin lies parallel to the membrane surface. In contrast, induction of the colicin channel by the membrane potential requires a profound reorganization of the helices association. These results are discussed in light of several proposed models of the membrane-bound colicin and channel structures.

Amino Acid Sequence↗
PubMed1993

A 76-amino acid disulfide loop in the Yersinia pseudotuberculosis invasin protein is required for integrin receptor recognition.

The Yersinia pseudotuberculosis invasin protein is a 986-amino acid protein that promotes bacterial penetration into mammalian cells by avidly binding multiple beta 1-chain integrins. A 192-amino acid carboxyl-terminal domain of invasin was previously shown to be sufficient for binding. Evidence is presented here that a 76-amino acid disulfide loop in the integrin binding domain of invasin is required for invasin-mediated cell binding and entry. Bacterial mutants that were altered at either of 2 cysteine residues in the binding domain of invasin were completely defective for entry. Purified invasin protein derivatives altered at either of these cysteines, in contrast to the wild-type invasin, did not promote either cell binding or penetration. Analysis of proteolytic products of invasin in the presence or absence of reducing agent provided evidence of an intra-chain disulfide bond near the carboxyl terminus of the protein. Alkylation of invasin derivatives with [3H]iodoacetate indicated that these 2 cysteines were normally disulfide-bonded. A treatment that resulted in the maximal reduction of the disulfide bond also resulted in maximal loss of cell attachment activity. These results indicate that the 76-amino acid disulfide loop at the carboxyl terminus of invasin is required for recognition by integrins.

Adhesins, Bacterial↗
PubMed1993

The secretory pathway is normal in dithiothreitol-treated cells, but disulfide-bonded proteins are reduced and reversibly retained in the endoplasmic reticulum.

Addition of 2 mM dithiothreitol (DTT) to HepG2 human hepatoma cells blocks secretion of newly made albumin and causes it to accumulate in the endoplasmic reticulum in the reduced state. Subsequent incubation of the cells in the absence of DTT allows the reduced albumin to form apparently normal disulfide bonds and to be secreted normally. Similarly, DTT treatment causes all newly made subunits of the H1 subunit of the asialoglycoprotein receptor to be retained in the endoplasmic reticulum in the reduced state; following removal of DTT H1 subunits form disulfide bonds via a normal endoplasmic reticulum folding intermediate and mature to the Golgi. Thus, apparently normal formation of disulfide bonds on these two proteins can occur post-translationally, as has been shown previously for the influenza HA hemagglutinin (Braakman, I., Helenius, J., and Helenius, A. (1992) EMBO J. 11, 1717-1722; Braakman, I., Helenius, J., and Helenius, A. (1992) Nature 356, 260-262). alpha 1-Antitrypsin contains no disulfide bonds; in the continuous presence of DTT it acquires a normal complement of complex oligosaccharides and is secreted at an only slightly reduced rate. Thus, the secretory pathway functions efficiently even when it is reduced by DTT. Endoplasmic reticulum retention of albumin and H1 during treatment with DTT presumably occurs because, without disulfide bonds, these proteins cannot fold properly, not because of any defect in the overall processes of vesicular transport and protein secretion.

Asialoglycoprotein Receptor↗
PubMed1976

Studies on the disulfide bonds of glycoprotein hormones. Course of reduction of bovine luteinizing hormone, bovine thyroid-stimulating hormone, and their subunits.

The five disulfide bonds of isolated alpha subunits of luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) are completely reduced at pH 8.5 in 15 min with no denaturant required and with only a slight excess of reducing agent. At pH 7.0, reduction is complete after 6 to 10 h. These results together with an earlier study concerning the positions of the two most readily reduced bonds (Cornell J.S., and Pierce, J.G. (1974) J. Biol. Chem. 249, 4166-4174) show that, in the isolated alpha subunit, all disulfides are readily accessible, although it is possible that a change in conformation, after rapid initial reduction of two disulfides, makes the remaining three more susceptible to reduction. No partially reduced and S-carboxymethylated intermediates were found at pH 7.0 other than those seen at pH 8.5, nor were additional intermediates found at pH 8.5 when reduction was initiated in the presence of alkylating agent. In contrast, reduction of the beta, hormone-specific, subunits of LH and TSH, while complete at pH 8.5 after 2 to 6 h, does not proceed to completion at pH 7.0 even after 24 h or upon addition of 6 M urea or large concentrations of reducing agent, and partially reduced intermediates useful in location of disulfide bridges can be trapped (e.g. Reeve, J.R., Cheng, K.-W., and Pierce, J.G. (1975) Biochem. Biophys. Res. Commun. 67, 149-155). Little or no reduction of the intact hormones is found at pH 7.0 in the absence of denaturing agents. This protection by the intact structure shows that the two most readily reduced disulfides of the alpha subunit and the single most readily reduced sidulfide of the beta subunits are either in regions of subunit-subunit contact or that these bonds become more reactive in the isolated subunits because of different influences by neighboring groups. At pH 8.5, intact LH is completely reduced after 6 h, but intact TSH is more resistant to reduction, which may reflect a higher affinity between subunits than exists in LH.

Animals↗
PubMed1994

The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation.

We have identified and functionally characterized a new Escherichia coli gene, dsbC, whose product is involved in disulfide bond formation in the periplasmic space. It corresponds to a previously sequenced open reading frame mapping upstream of recJ with no previously assigned function. Null mutations in dsbC were obtained using a screen for dithiothreitol (DTT)-sensitive mutants and were shown to result in the accumulation of reduced forms of a variety of disulfide bond-containing periplasmic proteins. This defect could be rescued by the addition of either oxidized DTT or cystine or by multicopy expression of dsbA, a known periplasmic disulfide oxidase. The DsbC protein is synthesized as a precursor form of 25.5 kDa which is processed to a 23.3 kDa mature species located in the periplasmic space. The DsbC protein was overexpressed, purified to homogeneity and shown to catalyse the reduction of insulin in a DTT-dependent manner at levels comparable with those of purified DsbA. The replacement of either cysteine residue of the predicted active site, F-(X4)-C-G-Y-C, completely inactivates DsbC protein function. We have further shown that in vivo overexpression of DsbC can functionally substitute for a loss of DsbA function. Taken together, all of our results demonstrate that DsbC acts in vivo as a disulfide oxidase.

Amino Acid Sequence↗
PubMed1993

Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli.

The redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione. The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue. From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 x 10(-4) M was determined, corresponding to a standard redox potential (E'0) of DsbA of -0.089 V. Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase. The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 x 10(-3) M to 3.9 x 10(-5) M between pH 4 and 8.5. The redox state-dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase "at work" during catalysis of oxidative protein folding.

Disulfides↗
PubMed1993

Charge-remote fragmentation in a disulfide-containing peptide, [Pen]-enkephalin, under fast atom bombardment collisionally activated dissociation conditions.

Fast atom bombardment collisionally activated dissociation tandem mass spectrometry (FAB CAD MS/MS) of a disulfide-containing peptide, [2-D-penicillamine, 5-D-penicillamine]- enkephalin ([Pen]-enkephalin), is described. Unlike those of most other disulfide-containing peptides investigated, CAD of the native, unreduced protonated molecule of [Pen]-enkephalin yields a relatively large number of fragment ions. Most of the peaks in the CAD spectrum represent fragmentations of the peptide backbone with 'unsymmetric' cleavage of the disulfide bond with charge retention on the N-terminus; the fragment ions generally do not contain a sulfur atom or a disulfide group. The CAD spectrum of the N-terminal ethyl-triphenylphosphonium derivative, on the other hand, shows few fragment ions and is dominated by the single peak at m/z 523. This dominant ion also results from 'unsymmetric' cleavage and corresponds to the analogous protonated species (m/z 235) in the spectrum of the underivatized peptide. The chemical method of charge localization by triphenylphosphonium derivative formation at one end of the peptide is shown to be useful for investigating fragmentation mechanisms in FAB CAD MS/MS. Comparison of the CAD spectra of derivatized and underivatized [Pen]-enkephalin suggests that charge-remote fragmentation plays a significant role in the high-energy dissociation of this disulfide-bonded peptide.

Enkephalin, D-Penicillamine (2,5)-↗
PubMed1994

Hydrogen exchange in BPTI variants that do not share a common disulfide bond.

Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30-51, 5-55, and 14-38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5-55]Ala and [30-51; 14-38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide-bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native-like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI folding intermediates containing either the 5-55 or the 30-51 disulfide bond.

Amides↗