The disulfide structure of denatured epidermal growth factor: preparation of scrambled disulfide isomers

The conformational stability of human epidermal growth factor (EGF) and the structure of denatured EGF were investigated using the technique of disulfide scrambling. Under denaturing conditions and in the presence of a thiol catalyst, the native EGF denatures by shuffling its three native disulfide bonds and converts to a mixture of scrambled isomers. Analysis by HPLC reveals that the denatured EGF is composed of about 10 fractions of scrambled isomers. The heterogeneity varies under different denaturing conditions, with the heat-denatured samples exhibiting the highest degree of heterogeneity. The disulfide structures of eight major scrambled isomers of EGF were determined. The most predominant isomer adopts the bead-form structure with disulfide bonds bridged by three pairs of neighboring cysteines: Cys⁶-Cys¹⁴, Cys²⁰-Cys³¹, and Cys³³-Cys⁴². The denaturation curve of EGF is determined by the relative yield of the scrambled and native species of EGF. EGF is a highly stable molecule and can be effectively denatured only by guanidine chloride at a concentration of greater than 4–5 M. At 8 M urea, less than 16% of the native EGF was denatured. The unusual conformational stability of EGF was compared with that of eight different disulfide proteins that were similarly characterized by the method of disulfide scrambling.     

Binding of calcium to anticoagulant protein S: role of the fourth EGF module

Protein S is an anticoagulant protein containing a Gla (enclosing gamma-carboxyglutamic acids) module, a TSR (thrombin sensitive region) module, four EGF (epidermal growth factor)-like modules, and a SHBG (sex hormone binding globulin)-like region. Protein S is a cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa but also has APC-independent activities. The function of the fourth EGF module (EGF4) in protein S has so far not been clear. We have now investigated this module through studies of recombinant wild-type protein S and a naturally occurring mutant (Asn217Ser). The mutant has essentially normal APC anticoagulant activity and a previously reported secretion defect. In the wild-type protein, Asn217 is normally beta-hydroxylated. The binding of calcium to wild-type protein S is characterized by four high-affinity binding sites with K(D) values ranging from 10(-)(7) to 10(-)(9) M. Three of these binding sites are located in EGF modules. Using surface plasmon resonance, competition with a calcium chelator, and antibody-based methods, we found that one high-affinity binding site for calcium was lost in protein S Asn217Ser but that the mutation also affected the calcium-dependent conformation of EGF1. We conclude that binding of calcium to EGF4 of protein S, involving Asn217, is important for the maintenance of the structure of protein S. Also, the abolition of binding of calcium to EGF4, related to Asn217, impairs both the structure and function of EGF1.     

Theoretical structural insights into the snakin/GASA family

Among the main classes of cysteine-stabilized antimicrobial peptides, the snakin/GASA family has not yet had any structural characterization. Through the combination of ab initio and comparative modeling with a disulfide bond predictor, the three-dimensional structure prediction of snakin-1 is reported here. The structure was composed of two long α-helices with a disulfide pattern of Cys^I-Cys^IX, Cys^II-Cys^VII, Cys^III-Cys^IV, Cys^V-Cys^XI, Cys^VI-Cys^XII and Cys^VIII-Cys^X. The overall structure was maintained throughout molecular dynamics simulation. Snakin-1 showed a small degree of structural similarity with thionins and α-helical hairpins. This is the first report of snakin-1 structural characterization, shedding some light on the snakin/GASA family.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Characterization of a Reduced Form of Plasma Plasminogen as the Precursor for Angiostatin Formation

Plasma plasminogen is the precursor of the tumor angiogenesis inhibitor, angiostatin. Generation of angiostatin in blood involves activation of plasminogen to the serine protease plasmin and facilitated cleavage of two disulfide bonds and up to three peptide bonds in the kringle 5 domain of the protein. The mechanism of reduction of the two allosteric disulfides has been explored in this study. Using thiol-alkylating agents, mass spectrometry, and an assay for angiostatin formation, we show that the Cys⁴⁶²-Cys⁵⁴¹ disulfide bond is already cleaved in a fraction of plasma plasminogen and that this reduced plasminogen is the precursor for angiostatin formation. From the crystal structure of plasminogen, we propose that plasmin ligands such as phosphoglycerate kinase induce a conformational change in reduced kringle 5 that leads to attack by the Cys⁵⁴¹ thiolate anion on the Cys⁵³⁶ sulfur atom of the Cys⁵¹²-Cys⁵³⁶ disulfide bond, resulting in reduction of the bond by thiol/disulfide exchange. Cleavage of the Cys⁵¹²-Cys⁵³⁶ allosteric disulfide allows further conformational change and exposure of the peptide backbone to proteolysis and angiostatin release. The Cys⁴⁶²-Cys⁵⁴¹ and Cys⁵¹²-Cys⁵³⁶ disulfides have −/+RHHook and −LHHook configurations, respectively, which are two of the 20 different measures of the geometry of a disulfide bond. Analysis of the structures of the known allosteric disulfide bonds identified six other bonds that have these configurations, and they share some functional similarities with the plasminogen disulfides. This suggests that the −/+RHHook and −LHHook disulfides, along with the −RHStaple bond, are potential allosteric configurations.     

Chemical synthesis of kurtoxin, a T-type calcium channel blocker

Kurtoxin isolated from the venom of scorpion, Parabuthus transvaalicus, is a 63-residue peptide with four intramolecular disulfide bonds which inhibits low-threshold T-type Ca²⁺channels. Kurtoxin was synthesized by native chemical ligation involving the coupling of (1--26)-thioester peptide and Cys²⁷-(28--63)-peptide. The former was synthesized by standard solid-phase peptide synthesis (SPPS) with Boc chemistry, while the latter was sequentially assembled from three protected segments onto a resin-bound C-terminal segment in a chloroform--phenol mixed solvent followed by deprotection reaction using HF. Each protected segment used for the coupling on a solid support was prepared on an N-[9-(hydroxymethyl)-2-fluorenyl] succinamic acid (HMFS) resin and detached from the resin by treatment with 20% Et ₃N in DMF to produce it in the form of an α-carboxylic acid. Synthetic kurtoxin obtained after the oxidative folding reaction was found to be identical with the natural product by means of several analytical procedures, and its disulfide structure was determined for the first time to be Cys¹²-Cys⁶¹, Cys¹⁶-Cys³⁷, Cys²³-Cys⁴⁴ and Cys²⁷-Cys⁴⁶ by peptide mapping, sequence analysis and mass measurements.     

Assignment of the three disulfide bonds in ShK toxin: A potent potassium channel inhibitor from the sea anemone Stichodactyla helianthus

Summary ShK toxin, a 35-residue peptide isolated from the Caribbean sea anemone Stichodactyla helianthus, is a potent inhibitor of the Kv 1.3 potassium channel in lymphocytes. The natural toxin contains three disulfide bonds. The disulfide pairings of the synthetic ShK toxin were elucidated as a prerequisite for studies on its structure-function relationships. The toxin was fragmented at pH 6.5 using either thermolysin or a mixture of trypsin and chymotrypsin followed by thermolysin. The fragments were isolated by RP-HPLC and were identified by sequence analysis and MALDI-TOF mass spectrometry. The three disulfides were unambiguously identified in either proteolytic digest: Cys³ to Cys³⁵, Cys¹² to Cys²⁸ and Cys¹⁷ to Cys³². The Cys³-Cys³⁵ disulfide, linking the amino- and carboxyl-termini, defines the characteristic cyclic structure of the molecule. A similar disulfide pairing motif is found in the snake venom-derived potassium channel blocker dendrotoxin and the mammalian antibiotic peptide defensins.     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

Sequential Comparison of Peptides Containing Half-cystine Residues from Ovalbumins of Six Avian Species

Hen ovalbumin contains one cystine disulfide (Cys⁷³-Cys¹²⁰) and four cysteine sulfhydryl groups (Cys¹¹,Cys³⁰,Cys³⁶⁷, and Cys³⁸²) in a single polypeptide chain of 385 amino acid residues. To investigate whether or not such a structure is shared by related avian species, the contents of disulfide-involved half-cystine residues and their positions in the primary structure of ovalbumins from five species were compared with those of hen ovalbumin. Ovalbumins were alkylated with a fluorescent dye, IAEDANS, under disulfide-reduced and disulfide-intact conditions and digested with a number of proteolytic enzymes. The sequences were deduced from peptides containing half-cystine residues labeled with the fluorescent dye. The results showed that the number of free cysteine sulfhydryl groups of ovalbumins was different among the species, three for guinea fowl and turkey (Cys¹¹, Cys³⁶⁷, and Cys³⁸²); and two for Pekin duck, mallard duck, and Emden goose (Cys¹¹ and Cys³³¹). On the other hand, a single intrachain disulfide bond could be identified from ovalbumins of five species using a combination of peptide mapping and N-terminal amino acid sequencing analysis under reduced and non-reduced conditions, in which the intrachain disulfide bond was like that of hen ovalbumin (Cys⁷³-Cys¹²⁰). The results also indicated that the variations in amino acid sequences on these peptides containing half-cystine residues bear a close relationship with the phylogeny of the six species.     

Assignment of the three disulfide Bridges of huwentoxin-I, a neurotoxin from the spiderSelenocosmia huwena

The positions of the disulfide bonds of huwentoxin-I, a neurotoxin from the spiderSelenocosmia huwena, have been determined. The existence of three disulfide bonds in the native toxin was demonstrated by mass spectroscopy and the lack of reactivity with a thiol reagent. The assignment procedure involved a combination of tryptic digestion of the native toxin and sequence analysis of both intact andin situ S-carboxymethylated toxin.In situ carboxymethylation is shown to be a useful procedure in sequencing of cysteine- and cystine-containing peptides. Sequence analysis of the intact, cross-linked toxin indicated that no amino acid phenylthiohydantoin (PTH) derivative is seen for the first half-cystine in a cross-linked pair, but that the PTH of dehydroalanine, which can be detected at 313 nm, is seen at the position of the second half-cystine. By sequencing disulfide cross-linked tryptic fragments, the three disulfide linkages in huwentoxin-I could be assigned as Cys₂-Cys₁₇, Cys₉-Cys₂₂, and Cys₁₆-Cys₂₉.     

Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase

Purified human glucocerebrosidase isolated from placenta was modified with [¹⁴C]-iodoacetic acid without reduction and digested with both protease-V8 at pH 4.0 followed by-chymotrypsin at pH 7.5. The majority of radioactivity was found in a peptide that contained the [¹⁴C]-carboxymethylated-cysteine identified as CM-Cys₁₈. Direct sequencing of the N-terminus of the intact labeled protein confirmed the modification of Cys₁₈. For identification of disulfide bond-containing peptides, another portion of glucocerebrosidase was alkylated with nonlabeled iodoacetic acid and then digested with protease V8 and-chymotrypsin as before. Twenty-eight HPLC fragments were collected. These purified peaks were then reduced with-mercaptoethanol followed by S-carboxymethylation with [¹⁴C]-iodoacetic acid. Three peptides among these 28 peptides generated two radioactive daughter peptides. These peptides were sequenced and the position of the radioactive CM-cysteines identified. The locations of these disulfides are Cys₄-Cys₁₆, Cys₂₃-Cys₃₄₂, and Cys₁₂₆-Cys₂₄₈. Attempts to reproduce the free sulfhydryl labeling experiments using the glucocerebrosidase isolated from Ceredase proved unsuccessful. No label was incorporated by this enzyme prior to reduction. This result suggests that the form of the protein used in the clinic differs from the native protein.     

Redox Regulation of Methionine Aminopeptidase 2 Activity

Protein translation is initiated with methionine in eukaryotes, and the majority of proteins have their N-terminal methionine removed by methionine aminopeptidases (MetAP1 and MetAP2) prior to action. Methionine removal can be important for protein function, localization, or stability. No mechanism of regulation of MetAP activity has been identified. MetAP2, but not MetAP1, contains a single Cys²²⁸-Cys⁴⁴⁸ disulfide bond that has an −RHStaple configuration and links two β-loop structures, which are hallmarks of allosteric disulfide bonds. From analysis of crystal structures and using mass spectrometry and activity assays, we found that the disulfide bond exists in oxidized and reduced states in the recombinant enzyme. The disulfide has a standard redox potential of −261 mV and is efficiently reduced by the protein reductant, thioredoxin, with a rate constant of 16,180 m⁻¹ s⁻¹. The MetAP2 disulfide bond also exists in oxidized and reduced states in glioblastoma tumor cells, and stressing the cells by oxygen or glucose deprivation results in more oxidized enzyme. The Cys²²⁸-Cys⁴⁴⁸ disulfide is at the rim of the active site and is only three residues distant from the catalytic His²³¹, which suggested that cleavage of the bond would influence substrate hydrolysis. Indeed, oxidized and reduced isoforms have different catalytic efficiencies for hydrolysis of MetAP2 peptide substrates. These findings indicate that MetAP2 is post-translationally regulated by an allosteric disulfide bond, which controls substrate specificity and catalytic efficiency.     

Human Eosinophil Major Basic Protein 2: Location of Disulfide Bonds and Free Sulfhydryl Groups

Eosinophil granule major basic protein 2 (MBP2 or major basic protein homolog) is a paralog of major basic protein (MBP1) and, similar to MBP1, is cytotoxic and cytostimulatory in vitro. MBP2, a small protein of 13,433 Da molecular weight, contains 10 cysteine residues. Mass spectrometry shows two cystine disulfide linkages (Cys²⁰–Cys¹¹⁵ and Cys⁹²–Cys¹⁰⁷) and 6 cysteine residues with free sulfhydryl groups (Cys², Cys²³, Cys⁴², Cys⁴³, Cys⁶⁸, and Cys⁹⁶). MBP2, similar to MBP1, has conserved motifs in common with C-type lectins. The disulfide bond locations are conserved among human MBP1, MBP2 and C-type lectins.     

Interplay between Disulfide Bonding and N-Glycosylation Defines SLC4 Na+-coupled Transporter Extracellular Topography

The extracellular loop 3 (EL-3) of SLC4 Na⁺-coupled transporters contains 4 highly conserved cysteines and multiple N-glycosylation consensus sites. In the electrogenic Na⁺-HCO₃⁻ cotransporter NBCe1-A, EL-3 is the largest extracellular loop and is predicted to consist of 82 amino acids. To determine the structural-functional importance of the conserved cysteines and the N-glycosylation sites in NBCe1-A EL-3, we analyzed the potential interplay between EL-3 disulfide bonding and N-glycosylation and their roles in EL-3 topological folding. Our results demonstrate that the 4 highly conserved cysteines form two intramolecular disulfide bonds, Cys⁵⁸³-Cys⁵⁸⁵ and Cys⁶¹⁷-Cys⁶⁴², respectively, that constrain EL-3 in a folded conformation. The formation of the second disulfide bond is spontaneous and unaffected by the N-glycosylation state of EL-3 or the first disulfide bond, whereas formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected by N-glycosylation. Importantly, EL-3 from each monomer is adjacently located at the NBCe1-A dimeric interface. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. This unique adjacent parallel location of two symmetrically folded EL-3 loops from each monomer resembles a domain-like structure that is potentially important for NBCe1-A function in vivo. Moreover, the formation of this unique structure is critically dependent on the finely tuned interplay between disulfide bonding and N-glycosylation in the membrane processed NBCe1-A dimer.     

Dimeric α-cobratoxin X-ray structure: localization of intermolecular disulfides and possible mode of binding to nicotinic acetylcholine receptors

In Naja kaouthia cobra venom, we have earlier discovered a covalent dimeric form of α-cobratoxin (αCT-αCT) with two intermolecular disulfides, but we could not determine their positions. Here, we report the αCT-αCT crystal structure at 1.94 Å where intermolecular disulfides are identified between Cys³ in one protomer and Cys²⁰ of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys²⁶ and Cys³⁰ in the central loops II, have the same positions as in monomeric α-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the αCT-αCT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the κ-bungarotoxin solution structure. Selective reduction of Cys²⁶-Cys³⁰ in one protomer does not affect the activity against the α7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents α7 nAChR recognition. On the contrary, reduction of one or both Cys²⁶-Cys³⁰ disulfides in αCT-αCT considerably potentiates inhibition of the α3β2 nAChR by the toxin. The heteromeric dimer of α-cobratoxin and cytotoxin has an activity similar to that of αCT-αCT against the α7 nAChR and is more active against α3β2 nAChRs. Our results demonstrate that at least one Cys²⁶-Cys³⁰ disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by α7 nAChR, although it is less important for interaction of covalent TFT dimers with the α3β2 nAChR.     

Three-dimensional structure of α-conotoxin EI determined by1H NMR spectroscopy

Summary α-conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom ofConus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organTorpedo, showing a novel selectivity profile when compared to other α-conotoxins. The 3D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between THr⁹ and Met¹² that is stabilized by the Cys⁴-Cys¹⁰ disulfide bond and turns involving Cys⁴-Cys⁵ and Asn¹⁴-Pro¹⁵. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other α4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other α4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to differences in the surface charge distribution among these α4/7-conotoxins. The implications for binding of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI. Supplementary material available:¹H resonance assignments of α-conotoxin EI.     

Calcium-binding EGF-like modules in coagulation proteinases: function of the calcium ion in module interactions

Epidermal growth factor (EGF)-like modules are involved in protein-protein interactions and are found in numerous extracellular proteins and membrane proteins. Among these proteins are enzymes involved in blood coagulation, fibrinolysis and the complement system as well as matrix proteins and cell surface receptors such as the EGF precursor, the low density lipoprotein receptor and the developmentally important receptor, Notch. The coagulation enzymes, factors VII, IX and X and protein C, all have two EGF-like modules, whereas the cofactor of activated protein C, protein S, has four EGF-like modules in tandem. Certain of the cell surface receptors have numerous EGF modules in tandem. A subset of EGF modules bind one Ca(2+). The Ca(2+)-binding sequence motif is coupled to a sequence motif that brings about beta-hydroxylation of a particular Asp/Asn residue. Ca(2+)-binding to an EGF module is important to orient neighboring modules relative to each other in a manner that is required for biological activity. The Ca(2+) affinity of an EGF module is often influenced by its N-terminal neighbor, be it another EGF module or a module of another type. This can result in an increase in Ca(2+) affinity of several orders of magnitude. Point mutations in EGF modules that involve amino acids which are Ca(2+) ligands result in the biosynthesis of biologically inactive proteins. Such mutations have been identified, for instance, in factor IX, causing hemophilia B, in fibrillin, causing Marfan syndrome, and in the low density lipoprotein receptor, causing hypercholesterolemia. In this review the emphasis will be on the coagulation factors.     

Allosteric Control of βII-Tryptase by a Redox Active Disulfide Bond

The S1A serine proteases function in many key biological processes such as development, immunity, and blood coagulation. S1A proteases contain a highly conserved disulfide bond (Cys¹⁹¹–Cys²²⁰ in chymotrypsin numbering) that links two β-loop structures that define the rim of the active site pocket. Mast cell βII-tryptase is a S1A protease that is associated with pathological inflammation. In this study, we have found that the conserved disulfide bond (Cys²²⁰–Cys²⁴⁸ in βII-tryptase) exists in oxidized and reduced states in the enzyme stored and secreted by mast cells. The disulfide bond has a standard redox potential of −301 mV and is stoichiometrically reduced by the inflammatory mediator, thioredoxin, with a rate constant of 350 m⁻¹ s⁻¹. The oxidized and reduced enzymes have different substrate specificity and catalytic efficiency for hydrolysis of both small and macromolecular substrates. These observations indicate that βII-tryptase activity is post-translationally regulated by an allosteric disulfide bond. It is likely that other S1A serine proteases are similarly regulated.     

Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme

A three-disulfide form of hen egg white lysozyme with Cys⁶ and Cys¹²⁷ blocked by carboxymethyl groups was prepared, purified, and characterized for eventual use in protein folding experiments. Trypsin digestion followed by proline-specific endopeptidase digestion facilitated the unambiguous assignment of the disulfide bond pairings and the modified residues in this derivative. 3SS-lysozyme demonstrated nearly full enzymatic activity at itspH optimum,pH 5.5. The 3SS-lysozyme derivative and unmodified lysozyme were shown to be identical by CD spectroscopy atpH 3.6. Immunochemical binding assays demonstrated that the conformation of lysozyme was perturbed predominantly only locally by breaking and blocking the disulfide bond between Cys⁶ and Cys¹²⁷. Both 3SS-lysozyme and unmodified lysozyme exhibited reversible thermally induced transitions atpH 2.0 but theT _m of 3SS-lysozyme, 18.9°C, was found to be 34° lower than that of native lysozyme under the same conditions. The conformational chemical potential of the denatured form of unmodified lysozyme was determined from the transition curves to be approximately 6.7 kcal/mol higher than that of the denatured form of 3SS-lysozyme, atpH 2.0 and 35°C, if the conformational chemical potential for the folded forms ofboth 3SS-lysozyme and unmodified lysozyme is arbitrarily assumed to be 0.0 kcal/mol. A calculation of the increase in the theoretical loop entropy of denatured 3SS-lysozyme resulting from the cleavage of the Cys⁶-Cys¹²⁷ disulfide bond, however, yielded a value of only 5.4 kcal/mol for the difference in conformational chemical potential. This suggests that, in addition to the entropic component, there is also an enthalpic contribution to the difference in the conformational chemical potential corresponding to approximately 1.3 kcal/mol. Thus, it is concluded that the reduction and blocking of the disulfide bond between Cys⁶ and Cys¹²⁷ destabilizes 3SS-lysozyme relative to unmodified lysozyme predominantly by stabilizing the denatured conformation by increasing its chain entropy.Cornell Biotechnology Army Research Office Predoctoral Fellow, 1986–1989.     

Assignment of the three disulfide Bridges of huwentoxin-I,a neurotoxin from the spiderSelenocosmia huwena

The positions of the disulfide bonds of huwentoxin-I, a neurotoxin from the spiderSelenocosmia huwena, have been determined. The existence of three disulfide bonds in the native toxin was demonstrated by mass spectroscopy and the lack of reactivity with a thiol reagent. The assignment procedure involved a combination of tryptic digestion of the native toxin and sequence analysis of both intact andin situ S-carboxymethylated toxin.In situ carboxymethylation is shown to be a useful procedure in sequencing of cysteine- and cystine-containing peptides. Sequence analysis of the intact, cross-linked toxin indicated that no amino acid phenylthiohydantoin (PTH) derivative is seen for the first half-cystine in a cross-linked pair, but that the PTH of dehydroalanine, which can be detected at 313 nm, is seen at the position of the second half-cystine. By sequencing disulfide cross-linked tryptic fragments, the three disulfide linkages in huwentoxin-I could be assigned as Cys₂-Cys₁₇, Cys₉-Cys₂₂, and Cys₁₆-Cys₂₉.