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PubMed2005

Characterization of the disulfide bonds and free cysteine residues of the Chlamydia trachomatis mouse pneumonitis major outer membrane protein.

Members of the genus Chlamydia lack a peptidoglycan layer. As a substitute for peptidoglycan, it has been proposed that several cysteine rich proteins, including the major outer membrane protein (MOMP), form disulfide bonds to provide rigidity to the cell wall. Alignment of the amino acids sequences of the MOMP from various serovars of Chlamydia showed that they have from 7 to 10 cysteine residues and seven of them are highly conserved. Which of these are free cysteine residues and which are involved in disulfide bonds is unknown. The complexity of the outer membrane of Chlamydia precludes at this point the characterization of the structure of the cysteines directly in the bacteria. Therefore, mass spectrometric analysis of a purified and refolded MOMP was used in this study. Characterization of the structure of this preparation of the MOMP is critical because it has been shown, in an animal model, to be a very effective vaccine against respiratory and genital infections. Here, we demonstrated that in this MOMP preparation four cysteines are involved in disulfide bonds, with intramolecular pairs formed between Cys(48) and Cys(55) and between Cys(201) and Cys(203). A stepwise alkylation, reduction, alkylation process using two different alkylating reagents was required to establish the Cys(48)-Cys(55) disulfide pair. The other residues in MOMP, Cys(51), Cys(136), Cys(226), and Cys(351), are free cysteines and could potentially form disulfide-linked complexes with other MOMP or other membrane proteins.

Amino Acid Sequence↗
PubMed2004

An engineered disulfide bond between residues 69 and 238 in extended-spectrum beta-lactamase Toho-1 reduces its activity toward third-generation cephalosporins.

Previous crystallographic structural analysis of extended-spectrum beta-lactamase Toho-1 predicted that the high flexibility of beta-strand B3, the region that contains a conserved KTG motif and forms one wall of the substrate-binding site, could be one of the key features contributing to Toho-1 activity toward third-generation cephalosporins. To investigate whether this possible flexibility really affects the substrate profile of this enzyme, two Toho-1 mutants have been produced, G238C and G238C/G239in, in which the glycine residue at position 238 was replaced with a cysteine and an additional glycine residue was inserted. Our intent was to introduce a disulfide bond between the cysteine residues at positions 69 and 238, and thus to lock the position of beta-strand B3. The results of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) titration indicated formation of a new disulfide bridge in the G238C mutant, although disulfide bond formation was not confirmed in the G238C/G239in mutant. Kinetic analysis showed that the activity of the G238C mutant decreased drastically against third-generation cephalosporins, while its catalytic efficiency against penicillins and first-generation cephalosporins was almost identical to that of the wild-type enzyme. This result was consistent with the prediction that flexibility in beta-strand B3 was critical for activity against third-generation cephalosporins in Toho-1. Furthermore, we have determined the crystal structure of the G238C mutant enzyme to analyze the structural changes in detail. The structural model clearly shows the introduction of a new disulfide bridge and that there is no appreciable difference between the overall structures of the wild-type enzyme and the G238C mutant, although the introduced disulfide bond slightly influenced the positions of Ser237 on beta-strand B3 and Asn170 on the Omega loop. The results of our kinetic and structural analyses suggest that the flexibility of beta-strand B3, as well as the positions of Ser237 and the Omega loop, is critical for the substrate specificity expansion of Toho-1.

Amino Acid Sequence↗
PubMed2005

Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations.

The engineered disulfide bridge A23C/L203C in human carbonic anhydrase II, inserted from homology modeling of Neisseria gonorrhoeae carbonic anhydrase, significantly stabilizes the native state of the protein. The inserted cysteine residues are placed in the interior of the structure, and because of the conformationally restrained localization, the protein is expressed in the reduced state and the cysteines are not readily oxidized. However, upon exposure to low concentrations of denaturant (0.6 M guanidine hydrochloride), corresponding to the lower part of the denaturation curve for the first unfolding transition, the oxidation rate of correctly formed disulfide bridges was markedly increased. By entropy estimations it appears that the increased flexibility, induced by the denaturant, enables the cysteines to find each other and hence to form the disulfide bridge. The outlined strategy of facilitating formation of disulfide bonds by addition of adjusted concentrations of a denaturant should be applicable to other proteins in which engineered cysteine residues are located in nonideal conformations. Moreover, a S99C/V242C variant was constructed, in which the cysteine residues are located on the surface. In this mutant the disulfide bridge was spontaneously formed and the native state was considerably stabilized (midpoint concentration of unfolding was increased from 1.0 to 1.4 M guanidine hydrochloride).

Alanine↗
PubMed2006

Evidence for activation of tissue factor by an allosteric disulfide bond.

Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF on the plasma membrane of cells resides mostly in a cryptic configuration, which rapidly transforms into an active configuration in response to certain stimuli. The extracellular part of TF consists of two fibronectin type III domains. The disulfide bond in the membrane proximal domain (Cys186-Cys209) is atypical for domains of this type in that it links adjacent strands in the same beta sheet, what we have called an allosteric bond. Ablation of the allosteric disulfide by mutating both cysteine residues severely impairs procoagulant activity. The thiol-alkylating agents N-ethylmaleimide and methyl methanethiolsulfonate block TF activation by ionomycin, while the thiol-oxidizing agent HgCl2 and dithiol cross-linkers promote activation. TF activation could not be explained by exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Cryptic TF contained unpaired cysteine thiols that were depleted upon activation, and de-encryption was associated with a change in the conformation of the membrane-proximal domain. These findings imply that the Cys186-Cys209 disulfide bond is reduced in the cryptic form of TF and that activation involves formation of the disulfide. It is likely that formation of this disulfide bond changes the conformation of the domain that facilitates productive binding of factors IX and X.

Blood Coagulation Factors↗
PubMed1996

Recombinant immunotoxin containing a disulfide-stabilized Fv directed at erbB2 that does not require proteolytic activation.

PE35/e23(dsFv)KDEL is a recombinant immunotoxin composed of a recombinant form of Pseudomonas exotoxin that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-erbB2 monoclonal antibody e23. In this molecule, the variable heavy (V H) domain is inserted near the carboxyl terminus of PE at position 607 and the variable light (V L) domain is connected to the V H domain by a disulfide bond engineered into the framework region. The disulfide bond forms between cysteines introduced at position 44 of V H and position 99 of V L [Reiter et al. (1994) J. Biol. Chem. 269, 18327-18331]. In contrast to other PE-derived Fv fusion proteins, this type of recombinant toxin does not need proteolytic activation of the toxin domain. PE35/e23(dsFv)KDEL is very cytotoxic toward erbB2 antigen-expressing N87 cells (IC50 = 0.8 ng/mL) despite the fact that it binds to the erbB2 protein only 25% as well as e23(dsFv)PE38KDEL, in which the dsFv moiety is located at the amino terminus of the toxin. The lower binding affinity is probably due to interference by domain III of PE with the amino terminus of e23(V H), possibly where the antigen binding sites are located. Nevertheless, the specificity of immunotoxin is still retained, and it is very stable at 37 degrees C. Because of its small size, stability, and activity without proteolytic processing, this immunotoxin may be advantageous for tumor treatment. PE35/e23(dsFv)KDEL was also used to gain information about whether reduction of the disulfide bonds connecting V H and V L occur in the endoplasmic reticulum (ER) or in a proximal compartment. To do this, we switched the ER retention sequence KDEL from the toxin--V H subunit to the V L subunit. Our results suggest that reduction of the disulfide bond connecting the dsFv heterodimer occurs before the immunotoxin reaches the ER, where translocation to the cytosol appears to occur.

ADP Ribose Transferases↗
PubMed1996

The plasma and cytoplasmic forms of human gelsolin differ in disulfide structure.

Gelsolin is a widely distributed actin binding protein that regulates actin filament length. It exists in both an intracellular and an extracellular form that is derived from a single gene by alternative splicing. Both forms contain the six homologous domains that are responsible for function. Little is known regarding differences between the forms. We have used a combination of cysteine-specific modification with 4-vinylpyridine, HPLC peptide mapping methods, and mass spectrometry to analyze the disulfide structures of human plasma and cytoplasmic gelsolin. Of the five Cys residues in the human gelsolin sequence, all were present in the free thiol form in human cytoplasmic gelsolin, while only three of them were free thiols in the human plasma form. Cys residues 188 and 201 in domain 2 of plasma gelsolin were disulfide linked. Recombinant human plasma gelsolin that had been expressed intracellularly in Escherichia coli and as a secreted protein from Cos green monkey cells was also investigated. The E. coli product lacked the disulfide but could be converted to the plasma-like structure with mild oxidation while the mammalian product formed the correct disulfide prior to isolation. Structural differences were also detected by limited proteolysis with plasmin. The differences in proteolytic susceptibility were also due to perturbations in domain 2. These studies demonstrate that the intracellular and extracellular gelsolins are structurally distinct and suggest that at least some of the preparations of recombinant gelsolin that are being used to study structure/function may be improperly folded. The experiments also demonstrate a general method for the location of disulfide bonds in proteins.

Actins↗
PubMed1996

Site-directed mutagenesis of conserved cysteine residues in porcine membrane dipeptidase. Cys 361 alone is involved in disulfide-linked dimerization.

Membrane dipeptidase (EC 3.4.13.19) is a glycosylphosphatidylinositol-anchored glycoprotein of the renal brush border which exists as a disulfide-linked homodimer. Porcine membrane dipeptidase has a subunit M(r) of 47 kDa, and the mature protein contains seven cysteine residues per subunit, six of which are conserved in the human enzyme. Chemical modification established that cysteine residues are not involved in enzyme activity. In order to determine which of the cysteine residues are involved in the interchain disulfide bond, we have used a site-directed mutagenesis approach. Each of the conserved cysteine residues was replaced by glycine or alanine. The single mutants (C71G, C93A, C154G, C226A, C258G, and C361G) were expressed in COS-1 cells and their enzymatic activity and oligomeric structure determined. Only the C361G mutant migrated as a polypeptide of 47 kDa when subjected to denaturing polyacrylamide gel electrophoresis under nonreducing conditions. Thus, cysteine 361 is the only residue involved in disulfide linkage between the subunits. This places the disulfide bond close to the site of GPI anchor addition (Ser 368 in the porcine enzyme) and to the membrane surface. Titration of the human and porcine proteins with 2-nitro-5-thiosulfabenzoate indicates that membrane dipeptidase additionally possesses two intrachain disulfide bonds. On native polyacrylamide gel electrophoresis, the C361G mutant migrates in a manner identical to that of the wild type, indicating that the protein remains associated as a noncovalent homodimer. The expressed C361G mutant, unlike the wild type, is released from COS-1 cell membranes by trypsin and by an endogenous serine protease.

Animals↗
PubMed1996

Folding of omega-conotoxins. 2. Influence of precursor sequences and protein disulfide isomerase.

The peptide Ca2+ channel antagonists found in the venoms of Conus snails, omega-conotoxins, are synthesized as precursors that include a leader peptide, presumed to direct the polypeptide to the endoplasmic reticulum, and a propeptide of unknown function. In addition, the precursors are synthesized with a C-terminal Gly residue that is posttranslationally converted to a terminal amide group. In order to determine whether the precursor sequences contain information that helps direct folding of the mature sequences, the disulfide-coupled folding of mature omega-conotoxin MVIIA was compared with that of two putative precursor forms: pro-omega-MVIIA-Gly, which contains the propeptide and the C-terminal Gly residue, and omega-MVIIA-Gly, which differs from the mature form only at the C-terminus. The three forms folded with similar kinetics, but the folding efficiency of omega-MVIIA-Gly was greater than 80%, versus approximately 50% for both mature omega-MVIIA and the form containing the propeptide. The enzyme protein disulfide isomerase was found to catalyze disulfide formation and folding of all three forms similarly. The affinity of omega-MVIIA-Gly for receptors in chick brain synaptosomes was approximately 10-fold lower than that of the mature peptide, and the N-terminal propeptide of pro-omega-MVIIA-Gly was found to decrease binding further, by approximately 100-fold. These results suggest that the omega-conotoxins do not rely on the propeptide region of their precursors to facilitate folding. Rather, the mature sequence contains most of the information required to specify the native disulfide pairings and three-dimensional conformation. The C-terminal Gly may enhance the folding efficiency by forming interactions that stabilize the native conformation with respect to other disulfide-bonded forms.

Amino Acid Sequence↗
PubMed1997

NMR solution structure of a two-disulfide derivative of charybdotoxin: structural evidence for conservation of scorpion toxin alpha/beta motif and its hydrophobic side chain packing.

The alpha/beta scorpion fold consisting of a short alpha-helix and beta-sheet is a structural motif common to scorpion toxins, insect defensins, and plant gamma-thionins that invariably contains three disulfides. CHABII is a two-disulfide derivative of the scorpion toxin charybdotoxin (ChTX), chemically synthesized by inserting two L-alpha-aminobutyric acids in place of the two half-cystine residues involved in the disulfide 13-33. This disulfide is one of the two disulfides which connect the alpha-helix to the beta-sheet. The solution structure of CHABII was determined at pH 6.3 and 5 degrees C using 2D NMR and simulated annealing from 513 distance and 46 dihedral angle constraints. The NMR structure of CHABII is well-defined as judged from the low value of the averaged backbone rms deviation between the 30 lowest energy structures and the energy-minimized mean structure ((rmsd) = 0.65 A for the entire sequence and 0.48 A for the segment 3-36). Analysis and comparison of the solution structures of CHABII and ChTX lead to the following conclusions: (i) the fold of CHABII is similar to that of ChTX as indicated by the low value of the averaged backbone atomic rms deviation between the 10 lowest energy solution structures of the two proteins (1.44 A); (ii) the packing of the hydrophobic core is well-preserved, underlying the critical structural role of the hydrophobic interactions even for such a small and cysteine-rich protein as ChTX.

Amino Acid Sequence↗
PubMed1998

Self-association of disulfide-dimerized melittin analogues.

Two cysteine substitutions of bee venom melittin have been synthesized to investigate the effects of disulfide cross-linking on the self-association properties of the peptide in solution. K23C melittin (mltK23C) was designed to link nonpolar surfaces of the amphipathic melittin helix on the basis of the close juxtaposition of pairs of K23 side chains in the crystal of the native melittin tetramer. K23Q/Q25C melittin (mltQ25C) was designed to link the polar surfaces of the peptide such that self-association in membrane bound states might be stabilized. The mltK23C disulfide dimer, (mltK23C)2, is highly structured at low pH under conditions where native melittin, and the mltK23C monomer, are unstructured. High-resolution NMR, circular dichroism, and fluorescence spectroscopy established that (mltK23C)2 is a helical monomer (pseudodimer) with stable helical segments between residues 2-13 and 15-25. Although the symmetrical nature of the pseudodimer prevented high-resolution structure determination, analysis of calculated hydrogen bond lengths, chemical shifts, near-UV circular dichroism, and urea denaturation demonstrated similarities with alpha-helical coiled coils and with the structure of native melittin in methanol. Stopped flow fluorescence showed that (mltK23C)2 underwent pH- and divalent anion-linked dimerization to a melittin-like pseudotetramer, indicating that a pair of disulfide bonds could be accommodated in a structure similar to the native melittin crystal structure. Despite incorporation of two disulfide bonds into the melittin tetramer, the folding free energy (DeltaGw) of [(mltK23C)2]2 was similar to that for the native melittin tetramer under the condition used. Incorporation of a disulfide bond on the polar helix face in melittin did not stabilize helical structure in the absence of self-association. Instead, this molecule underwent pH- and divalent anion-linked self-association to an ill-defined aggregate which precipitated.

Amino Acid Sequence↗
PubMedDate not supplied

Cleavage of disulfide-linked fetuin-bisphosphonate conjugates with three physiological thiols.

An effective therapeutic agent for treatment of bone diseases is expected to exhibit a high affinity to bone. Conjugating proteins to bisphosphonates (BPs), a class of molecules with an exceptional affinity to bone mineral hydroxyapatite (HA), is a feasible means to impart such a bone affinity. Protein-BP conjugates with cleavable linkages, which allow protein release from the mineral, are preferable over conjugates with stable linkages. To this end, 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (thiolBP) was conjugated onto fetuin, a model protein, using N-succinimidyl-3-(2-pyridyldithio)propionate to create disulfide-linked conjugates. Although the fetuin-thiolBP conjugates were stable under aqueous conditions, the disulfide linkage was readily cleaved in the presence of the physiological thiols l-cysteine, dl-homocysteine, and l-glutathione. dl-Homocysteine exhibited the highest cleavage of the disulfide linkage among these thiols. The imparted bone affinity as a result of thiolBP conjugation, as assessed by HA binding in vitro, was eliminated upon cleavage of the disulfide linkage. The cleavage of the conjugates bound to HA was as effective as the conjugate cleavage in solution, and even more so at high concentrations of l-glutathione. In conclusion, disulfide-linked fetuin-thiolBP conjugates exhibited a high affinity to HA, which was readily lost upon cleavage with thiols found in physiological milieu.

Biocompatible Materials↗
PubMed2006

Disulfide cross-linked polymer capsules: en route to biodeconstructible systems.

Hydrogen-bonded multilayer thin films were constructed using poly(vinylpyrrolidone) and poly(methacrylic acid) functionalized with cysteamine. The resulting films included thiol moieties that were cross-linked to render the films stable at physiological pH. Film buildup was followed using quartz crystal microgravimetry, which was also used to demonstrate the improved stability imparted by reacting the thiol moieties to form disulfide bonds. Films without disulfide bonds were readily deconstructed at physiological pH, while those with disulfide bonds were swollen upon exposure to this pH (7) but remained intact. Addition of a common thiol-disulfide exchange reagent, dithiothreitol (DTT) at pH 7 led to disassembly of the multilayer films. The films were also prepared on colloidal substrates (as demonstrated using confocal microscopy) and were used to retain a model drug (fluorescently labeled transferrin) and release this molecule when triggered by the addition of DTT. This approach has potential for the in vivo applications of hollow capsules, as thiol-disulfide exchange leading to deconstruction of the capsules can occur with the assistance of intracellular proteins.

Biodegradation, Environmental↗
PubMed2006

Thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive degradable polymer.

We report a reverse thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive biodegradable polymer. The poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) aqueous solutions studied in this research underwent sol-gel-sol or sol-gel-sol-gel transition depending on the molecular weight and concentration of the polymer, whereas the corresponding disulfide multiblock copolymer aqueous solutions underwent sol-gel transition as the temperature increased in a range of 0-60 degrees C. The hydrophobic dye solubilization and dynamic light scattering of the polymer aqueous solution suggest that the poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)s undergo unimer (3 nm) to micelle (12 nm) transition, whereas the disulfide multiblock copolymers undergo unimer (6 nm) to aggregated polymer (600 nm) transition as the temperature increases. The gel duration increased from 6 h (poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)) to more than 12 days (the corresponding disulfide multiblock copolymer) in phosphate buffer saline, and the gel duration of the latter depended on the glutathione concentration of the medium. The model drug, paclitaxel, was released from the in-situ-formed poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer gel in a glutathione concentration-sensitive manner.

Biocompatible Materials↗
PubMedDate not supplied

Role of aromacity, ring strain, and stereochemistry of selected disulfides and their congeners in the oxidative linkage of DNA strands at the major groove. A computational study.

Three aromatic disulfides and their two selected congeners were studied in order to see if they could be initiators or catalysts in a process that aims to tie together the two strands of a DNA double helix. Thermodynamic functions were calculated from first principle molecular computations on deoxyribose models and the disulfide congener. The gas reaction of one of the disulfides turned out to be the closest to the thermoneutral process. Solvation study caused a relatively minor energetic modification. The disulfides were subjected to docking studies, and the stabilities of the complexes obtained were further analyzed by molecular dynamics simulations. Only one of the two atropic disulfide enantiomers was the matching partner of the chiral DNA double helix, and therefore this remained in the major groove of the DNA. The other enantiomer dissociated from the major groove as it was incapable of forming a stable complex with the chiral double helix. A mechanistic scheme for the reaction has been investigated.

Catalysis↗
PubMed2002

Development of a novel method to populate native disulfide-bonded intermediates for structural characterization of proteins: implications for the mechanism of oxidative folding of RNase A.

RNase A, a model protein for oxidative folding studies, has four native disulfide bonds. The roles of des [40-95] and des [65-72], the two native-like structured three-disulfide-bonded intermediates populated between 8 and 25 degrees C during the oxidative folding of RNase A, are well characterized. Recent work focuses on both the formation of these structured disulfide intermediates from their unstructured precursors and on the subsequent oxidation of the structured species to form the native protein. The major obstacles in this work are the very low concentration of the precursor species and the difficulty of isolating some of the structured intermediates. Here, we demonstrate a novel method that enables the native disulfide-bonded intermediates to be populated and studied regardless of whether they have stable structure and/or are present at low concentrations during the oxidative folding or reductive unfolding process. The application of this method enabled us to populate and, in turn, study the key intermediates with two native disulfide bonds on the oxidative folding pathway of RNase A; it also facilitated the isolation of des [58-110] and des [26-84], the other two native-like structured des species whose isolation had thus far not been possible.

Animals↗
PubMed2003

Evaluation of multivalent dendrimers based on melamine: kinetics of thiol-disulfide exchange depends on the structure of the dendrimer.

The rate of thiol-disulfide exchange of dansyl groups mediated by dithiothreitol depends on the structure of the dendrimer. In general, the rate of exchange decreases as the size of the dendrimer increases. Dendrimers with disulfides attached near the core undergo exchange more slowly than dendrimers with disulfides near the periphery. Exchange is a bimolecular (noncooperative) process between dansyl-linked disulfides and dithiothreitol. No evidence for intramolecular macrocylization (cooperative) exchange is observed. Mass spectrometry is used to follow exchange in two dendrimers, providing qualitative and quantitative information about this process. Mathematical models suggest that the rates for exchange for all disulfides of a dendrimer are similar, but increase as the exchange reaction progresses.

Computer Simulation↗
PubMed2002

Heat-induced covalent complex between casein micelles and beta-lactoglobulin from goat's milk: identification of an involved disulfide bond.

Goat milk is characterized by a very low heat stability that could be attributed, in part, to the covalent interaction between whey proteins and casein micelles. However, the formation of such a complex in goat milk has never been evidenced. This study was designed to assess whether heat-induced covalent interaction occurs between purified casein micelles and beta-lactoglobulin. We used a multiple approach of ultracentrifugation of heated mixture, chromatographic fractionation of resuspended pellets, sequential enzyme digestion of disulfide-linked oligomers, and identification of disulfide-linked peptides by on-line liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), and tandem MS. We identified three different types of disulfide links: (1) expected intermolecular bridges between beta-Lg molecules; (2) disulfide bond involving two kappa-casein molecules; and (3) a disulfide bond between two peptides, one from beta-Lg and the other from kappa-casein. The involved sites in this last bond were Cys(160) of beta-Lg and Cys(88) of kappa-casein. Although the identified heterolinkage is possibly only one of several different types, the results of this study constitute the first direct evidence of the formation of a covalent complex between casein micelles and beta-lactoglobulin derived from goat milk.

Animals↗
PubMed2003

Crystal structures and structural stabilities of the disulfide bond-deficient soybean proglycinin mutants C12G and C88S.

The constituent subunits of seed storage protein 11S globulin have two disulfide bonds that are common among 11S globulins from legume and nonlegume seeds. In the case of the A1aB1b subunit of soybean 11S globulin, glycinin, Cys12-Cys45 and Cys88-Cys298 are observed by X-ray crystallography. The significance of these two disulfide bonds for structural stability was investigated by mutagenesis of Cys12 to Gly and of Cys88 to Ser. The disulfide bond-deficient mutants C12G and C88S could form the correct conformations identical to that of the wild-type proglycinin except in the vicinities of the mutation sites C12 and C88 as shown by their crystal structures. Thermal stability monitored by differential scanning calorimetry of the mutants indicated that the contribution of these disulfide bonds to the thermal stability of proglycinin A1aB1b is low, although there is a small difference in the extent of the contribution between the two disulfide bonds (Cys12-Cys45 > Cys88-Cys298). The contribution of Cys88-Cys298 to the resistance of proglycinin A1aB1b to proteinase digestion is higher than that of Cys12-Cys45. Possible effects of structure on the different properties of C12G and C88S are discussed.

Amino Acid Sequence↗