Disulfide bonds in neurotoxin-III from the sea anenome Radianthus macrodactylus

Positions of the three disulfide bridges in neurotoxin-III (RTX-III) from sea anemone Radianthus macrodactylus were determined: Cys3--Cys43, Cys5--Cys33, Cys26--Cys44. The cystine-containing peptides obtained by the staphylococcal proteinase/trypsin digestion of the intact RTX-III were investigated.     

Determination of the disulfide structure of sillucin, a highly knotted, cysteine-rich peptide, by cyanylation/cleavage mass mapping

The disulfide structure of sillucin, a highly knotted, cysteine-rich, antimicrobial peptide, isolated from Rhizomucor pusillus, has been determined to be Cys2--Cys7, Cys12--Cys24, Cys13--Cys30, and Cys14--Cys21 by disulfide mass mapping based on partial reduction and CN-induced cleavage enabled by cyanylation. The denatured 30-residue peptide was subjected to partial reduction by tris(2-carboxyethyl)phosphine hydrochloride at pH 3 to produce a mixture of partially reduced sillucin species; the nascent sulfhydryl groups were immediately cyanylated by 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate. The cyanylated species, separated and collected during reversed phase high-performance liquid chromatography, were treated with aqueous ammonia, which cleaved the peptide chain on the N-terminal side of cyanylated cysteine residues. The CN-induced cleavage mixture was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry before and after complete reduction of residual disulfide bonds in partially reduced and cyanylated species to mass map the truncated peptides to the sequence. Because the masses of the CN-induced cleavage fragments of both singly and doubly reduced and cyanylated sillucin are related to the linkages of the disulfide bonds in the original molecule, the presence of certain truncated peptide(s) can be used to positively identify the linkage of a specific disulfide bond or exclude the presence of other possible linkages.     

Disulfide bond assignment, lipid transfer activity and secondary structure of a 7-kDa plant lipid transfer protein, LTP2.

The 7-kDa lipid transfer proteins, LTP2s, share some amino-acid sequence similarities with the 9-kDa isoforms, LTP1s. Both proteins display an identical cysteine motif and, in this regard, LTP2s have been classified as lipid transfer proteins. However, in contrast with LTP1s, no data are available on their structure, cysteine pairings, lipid transfer and lipid binding properties. We reported on the isolation of two isoforms of 7-kDa lipid transfer protein, LTP2, from wheat seeds and showed for the first time that they indeed display lipid transfer activity. Trypsin and chymotrypsin digestions of the native LTP2 afforded the sequence of both isoforms and assignment of disulfide bonds. The cysteine pairings, Cys10--Cys24, Cys25--Cys60, Cys2--Cys34, Cys36--Cys67, revealed a mismatch at the Cys34-X-Cys36 motif of LTP2 compared to LTP1. Moreover, the secondary structure as determined by circular dichroism suggested an identical proportion of alpha helices, beta sheets and random coils. By analogy with the structure of the LTP1, we discussed what structural changes are required to accommodate the LTP2 disulfide pattern.     

Positions of disulfide bonds in rye (Secale cereale) seed chitinase-a

The positions of disulfide bonds of rye seed chitinase-a (RSC-a) were identified by the isolation of disulfide-containing peptides produced with enzymatic and/or chemical cleavages of RSC-a, followed by sequencing them. An unequivocal assignment of disulfide bonds in this enzyme was as follows: Cys3-Cysl8, Cys12-Cys24, Cys15-Cys42, Cys17-Cys31, and Cys35-Cys39 in the chitin-binding domain (CB domain), Cys82-Cys144, Cys156-Cys164, and Cys282-Cys295 in the catalytic domain (Cat domain), and Cys263 was a free form.     

Location of major antigenic sites of the beta subunit of human chorionic gonadotropin.

Purified beta subunit of human chorionic gonadotropin (hCG) was partially reduced with beta-mercaptoethanol, carboxymethylated, and digested with chymotrypsin. The peptides were isolated by high-voltage electrophoresis and paper chromatography. Five major disulfide-containing peptides were isolated, and their location in the parent molecule was established by amino acid composition and amino- and carboxy-terminal analyses. All of these peptides inhibited the binding of 125I-labeled hCG by anti-beta hCG serum. The inhibitory effect of these peptides was lost when their disulfide bonds were reduced and alkylated. Synthetic carboxy-terminal peptides were not inhibitory. Based on these data it is concluded that a major antigenic site of hCG resides in the region of residues 21-23 with a disulfide bond connecting cysteine-23 or -26 with the cysteines at positions 72 or 110.     

Assignment of all four disulfide Bridges in echistatin

Echistatin is a 49-amino-acid protein fromEchis carinatus venom. It contains four disulfide bonds. Since the disulfide bonding is critical for biological activity, it is very important to assign the disulfide linkage in this protein. Echistatin was incubated in 250 mM oxalic acid at 100°C for 4 hr under nitrogen. Under these conditions, many overlapping disulfide-containing peptides were identified by ionspray mass spectrometry. Ionspray MS/MS data indicate that the four disulfide bonds are Cys 2–Cys 11, Cys 7–Cys 32, Cys 8–Cys 37, and Cys 20–Cys 39. To our knowledge, this is the first time all four disulfide bonds in echistatin have been assigned in one experiment without disulfide bond exchange. This approach, which combines oxalic acid hydrolysis and ionspray MS/MS, may be very useful for assigning disulfide bridges in other proteins from the disintegrin family.     

Location of the disulfide bonds in the antitumor protein neocarzinostatin.

Two disulfide bonds in the antitumor antibiotic neocarzinostatin were determined chemically. The peptic and peptic/thermolytic peptides from the native protein were isolated by gel filtration and ion-exchange chromatography followed by reverse-phase HPLC. The cystine peptides obtained were oxidized separately by performic acid treatment and further separated by HPLC into cysteic acid peptides. Sequence analyses of the isolated peptides revealed the location of the disulfide bonds at Cys37-Cys47 and Cys88-Cys93.     

Assignment of disulfide bonds in proteins by partial acid hydrolysis and mass spectrometry

A new method is described for locating disulfide bonds in proteins which cannot be cleaved between half-cystinyl residues by enzymic methods, as is often the case for tightly coiled proteins, or for proteins in which half-cystinyl residues are not separated by residues required for enzymic cleavage. Partial acid hydrolysis of a model protein, hen egg-white lysozyme, produces a mixture of disulfide-containing peptides from which the disulfide connections may be deduced. The usefulness of a combination of HPLC, fast atom bombardment mass spectrometry, and computer-assisted analysis to identify disulfide-containing peptides present in the partial acid hydrolysate of the model protein is demonstrated. Chromatographic fractions of the hydrolysate were analyzed by mass spectrometry before and after chemical reduction of the disulfide bonds to determine the molecular weights of disulfide-containing peptides. Computer-assisted analysis was then used to relate the molecular weights of these peptides to specific segments of the protein from which the disulfide connectivities could be determined. Partial acid hydrolysis of proteins, which is attractive because it proceeds relatively independent of the amino acid sequence and structure, and because disulfide interchange is unlikely to occur in dilute acid, has become practical because disulfide-containing peptides present in complex mixtures can be identified rapidly and definitively by this method.     

Determination of the positions of the disulfide bonds in aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1

Aqualysin I is a heat-stable alkaline serine protease produced by Thermus aquaticus YT-1. Aqualysin I comprises 281 amino acid residues and contains four cysteine residues. The cysteine residues seemed to form disulfide bonds in the molecule. Thus, the positions of the disulfide bonds were investigated. Disulfide bond-containing peptides were identified by peptide mapping with HPLC before and after carboxymethylation of chymotryptic peptides of aqualysin I. The disulfide bond-containing peptides were isolated and then carboxymethylated. Carboxymethylcysteine-containing peptides were purified, and their amino acid compositions and sequences were determined. Based on the data obtained and the primary structure of aqualysin I, it was concluded that two disulfide bonds were formed between Cys67 and Cys99, and between Cys163 and Cys194.     

Efficient approach to synthesis of two-chain asymmetric cysteine analogs of receptor-binding region of transforming growth factor-alpha.

The putative receptor-binding region of human transforming growth factor-alpha (TGF alpha) has been shown to be contributed by two fragments: an A-chain (residue 12-18) and a 17-residue carboxyl fragment (residue 34-50) that includes a disulfide-containing C-loop (residue 34-43). An approach to the synthesis of two-chain analogs containing an intermolecular disulfide linked A-chain and the 17-residue carboxyl fragment (C-fragment) possessing receptor-binding activity is described. The synthesis was achieved by the solid-phase method using the Boc-benzyl protecting group strategy. The single Cys of the A-chain was activated as a mixed disulfide with 2-thiopyridine to form the intermolecular disulfide bond with Cys41 or Cys46 of the C-fragment on the resin support. Prior to this reaction, the acetamido (Acm) protecting group of Cys41 or Cys46 was removed by Hg(OAc)2 on the resin support. The peptide and side chain protecting groups including the S-methylbenzyl moiety of the Cys34 and Cys43 were concomitantly cleaved by high HF. The intramolecular disulfide with two unprotected Cys was formed in the presence of an intermolecular disulfide. This intramolecular disulfide bond formation was usually not feasible under the traditionally-held scheme at basic pH since disulfide interchange would occur faster than intramolecular oxidation. To prevent the disulfide interchange, a new method was devised. The intramolecular disulfide bond oxidation was mediated by dimethylsulfoxide at an acidic pH, at which the disulfide interchange reaction was suppressed. The desired product was obtained with a 60-70% yield.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of a mutant human lysozyme with a substituted disulfide bond

The three-dimensional structure of a mutant human lysozyme, W64CC65A, in which a non-native disulfide bond Cys64--Cys81 is substituted for the Cys65--Cys81 of the wild type protein by replacing Trp64 and Cys65 with Cys and Ala, respectively, was determined by X-ray crystallography and refined to an R-value of 0.181, using 33,187 reflections at 1.87-A resolution. The refined model of the W64CC65A protein consisted of four molecules, which were related by two noncrystallographic twofold axes and a translation vector. Although no specific structural differences could be observed among these four molecules, the overall B-factors of each molecule were quite different. The overall structure of W64CC65A, especially in the alpha-helical domain, was found to be quite similar to that of the wild type protein. Moreover, the side-chain conformation of the newly formed Cys64--Cys81 bond was quite similar to that of the Cys65--Cys81 bond of the wild-type protein. However, in the beta-sheet domain, the main-chain atoms of the loop region from positions 66-75 could not be determined, and significant structural changes due to the formation of the non-native disulfide bond could be observed. From these results, it is clear that the loop region of the mutant protein does not fold with the specific folding as observed in the wild-type protein.     

Assignment of the Disulfide Bridges in Bothropstoxin-I, a Myonecrotic Lys49 PLA2 Homolog from Bothrops jararacussu Snake Venom

Bothropstoxin-I (BthTX-I), a Lys49 phospholipase A₂ homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV₈) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV₈ digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-I disulfide bonds follow the normal pattern of group II PLA₂s.     

Assignment of the three disulfide bonds of Selenocosmia huwena lectin-I from the venom of spider Selenocosmia huwena.

The positions of the disulfide bonds of Selenocosmia huwena lectin-I (SHL-I) from the venom of the Chinese bird spider S. huwena have been determined. The existence of three disulfide bonds in the native SHL-I was proved by matrix-assisted laser desorption ionization time-of-flight mass spectroscopic analysis. To map the disulfide bonds, native SHL-I was proteolytically digested. The resulting peptides were separated by reverse phase high-performance liquid chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopic analysis indicated the presence of one disulfide bond Cys7-Cys19. The partially reduced peptides by using Tris-(2-carboxyethyl)-phosphine at pH 3.0 were purified by reverse phase high-performance liquid chromatography. Four M Guanidine-HCl was found to increase the yields of partially reduced peptides prominently. The free thiols were carboxamidomethlate by iodoacetamide. The specific location of another disulfide bond Cys2-Cys14 was proved by comparing N-terminal sequencing analysis of the partially reduced and alkylated SHL-I with that of the intact peptide. Finally, the three disulfide linkage of SHL-I could be assigned as Cys2-Cys14, Cys7-Cys19, Cys13-Cys26.     

Identification of the disulfide bonds in human plasma protein SP-40,40 (apolipoprotein-J).

SP-40,40, a human plasma protein, is a modulator of the membrane attack complex formation of the complement system as well as a subcomponent of high-density lipoproteins. In the present study, the positions of the disulfide bonds in SP-40,40 were determined. SP-40,40 was purified from human seminal plasma by affinity chromatography using an anti-SP-40,40 monoclonal antibody and reversed-phase, high-performance liquid chromatography (HPLC). The protein was digested with trypsin and the fragments were separated by reversed-phase HPLC. The peptides containing disulfide bonds were fluorophotometrically detected with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The peptides containing more than two disulfide bonds were further digested with Staphylococcus aureus V8 protease and lysylendopeptidase, and the fragments were isolated by HPLC. The amino acid compositions and the amino acid sequences of the peptides containing only a disulfide bond were determined. Disulfide bonds thus determined were between Cys58(alpha)-Cys107(beta), Cys68(alpha)-Cys99(beta), Cys75(alpha)-Cys94(beta), and Cys86(alpha)-Cys80(beta). Since there was no free sulfhydryl groups in the SP-40,40 molecule, Cys78(alpha) and Cys91(beta) should also be linked by a disulfide bond. It is notable that all of the disulfide bonds in SP-40,40 are not only formed by inter-chain pairing, but also appear to form an antiparallel ladder-like structure between the two chains. The unique structure could be related to the functions of SP-40,40.     

Stability and structure-forming properties of the two disulfide bonds of alpha-conotoxin GI

alpha-Conotoxin GI is a 13 residue snail toxin peptide cross-linked by Cys2-Cys7 and Cys3-Cys13 disulfide bridges. The formation of the two disulfide bonds by thiol/disulfide exchange with oxidized glutathione (GSSG) has been characterized. To characterize formation of the first disulfide bond in each of the two pathways by which the two disulfide bonds can form, two model peptides were synthesized in which Cys3 and Cys13 (Cono-1) or Cys2 and Cys7 (Cono-2) were replaced by alanines. Equilibrium constants were determined for formation of the single disulfide bonds of Cono-1 and Cono-2, and an overall equilibrium constant was measured for formation of the two disulfide bonds of alpha-conotoxin GI in pH 7.00 buffer and in pH 7. 00 buffer plus 8 M urea using concentrations obtained by HPLC analysis of equilibrium thiol/disulfide exchange reaction mixtures. The results indicate a modest amount of cooperativity in the formation of the second disulfide bond in both of the two-step pathways by which alpha-conotoxin GI folds into its native structure at pH 7.00. However, when considered in terms of the reactive thiolate species, the results indicate substantial cooperativity in formation of the second disulfide bond. The solution conformational and structural properties of Cono-1, Cono-2, and alpha-conotoxin GI were studied by 1H NMR to identify structural features which might facilitate formation of the disulfide bonds or are induced by formation of the disulfide bonds. The NMR data indicate that both Cono-1 and Cono-2 have some secondary structure in solution, including some of the same secondary structure as alpha-conotoxin GI, which facilitates formation of the second disulfide bond by thiol/disulfide exchange. However, both Cono-1 and Cono-2 are considerably less structured than alpha-conotoxin GI, which indicates that formation of the second disulfide bond to give the Cys2-Cys7, Cys3-Cys13 pairing induces considerable structure into the backbone of the peptide.     

Combined use of ESI-MS and UV diode-array detection for localization of disulfide bonds in proteins: application to an alpha-L-fucosidase of pea.

A simplified strategy is described for the assignment of disulfide bonds in proteins of medium to high molecular mass (10-30 kDa). The method combines the use of high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) and HPLC with UV diode-array detection (HPLC diode array). The denatured protein is subjected to proteolysis and the peptide mixture is divided into three fractions: (i) underivatized peptides, (ii) ethylpyridylated peptides, and (iii) reduced and ethylpyridylated peptides. The three peptide ensembles are then subjected to chromatographic and spectroscopic analysis. A systematic methodology is described to analyze the large amount of data obtained. The method was applied to the localization of disulfide bonds in alpha-L-fucosidase from pea. The two disulfide bonds were located between residues Cys64 and Cys109 and between Cys162 and Cys169, while Cys127 was free.     

Primary structure and disulfide bridge location of arrowhead double-headed proteinase inhibitors.

Two arrowhead proteinase inhibitors (inhibitors A and B) were characterized and their primary structures were determined. Both inhibitors A and B are double-headed and multifunctional protease inhibitors. Inhibitor A inhibits an equimolar amount of trypsin and chymotrypsin simultaneously and weakly inhibits kallikrein. Inhibitor B inhibits two molecules of trypsin simultaneously and inhibits kallikrein more strongly than does inhibitor A. The amino acid sequences of inhibitors A and B were determined by sequencing the reduced and S-carboxamidomethylated proteins and their peptides produced by cyanogen bromide or proteolytic lysylendopeptidase or Staphylococcus aureus V8 protease cleavage. Inhibitors A and B consist of 150 amino acid residues with three disulfide bonds (Cys 43-Cys 89, Cys 110-Cys 119, and Cys 112-Cys 115) and share 90% sequence identity, with 13 different residues. Since the primary structures are totally different from those of all other serine protease inhibitors so far known, these inhibitors might be classified into a new protease inhibitor family.     

Structure of an antifreeze polypeptide from the sea raven. Disulfide bonds and similarity to lectin-binding proteins.

The antifreeze polypeptide (AFP) from the sea raven, Hemitripterus americanus, is a member of the cystine-rich class of blood antifreeze proteins which enable survival of certain fishes at sub-zero temperatures. Sea raven AFP contains 129 residues with 10 half-cystine residues. We have analyzed these half-cystine residues and established that all 10 of the half-cystine residues appeared to be involved in disulfide bond formation and that disulfide bonds linked Cys7 to Cys18, Cys35 to Cys125, and Cys89 to Cys117. These assignments were established by extensive proteolytic digestions of native AFP using pepsin and thermolysin and purification of the peptides by Sephadex G-15 gel filtration chromatography, anion exchange chromatography, and C18 reverse-phase high performance liquid chromatography. Cystine-containing peptides were detected by a colorimetric assay using nitrothiosulfobenzoate. Disulfide-containing peptides were reduced and alkylated, purified, and analyzed by amino acid analysis. The unreduced disulfide-linked peptides were sequenced directly by automated Edman degradations to confirm the disulfide assignments. Possible arrangements of the two remaining disulfide bonds include linkages Cys69/111 to Cys100/101. The sea raven AFP shares structural similarity with pancreatic stone protein and several lectin-binding proteins, especially with respect to half-cystines, glycines, and bulky aromatic residues. Two of the disulfide linkages we determined for sea raven AFP: Cys7-Cys18 and Cys35-Cys125, are conserved in these proteins. These similarities in covalent structure suggest that the sea raven AFP, pancreatic stone protein, and several lectin-binding proteins comprise a family of proteins which may possess a common fold.     

2,2′‐Dipyridyl diselenide: A chemoselective tool for cysteine deprotection and disulfide bond formation

There are many examples of bioactive, disulfide‐rich peptides and proteins whose biological activity relies on proper disulfide connectivity. Regioselective disulfide bond formation is a strategy for the synthesis of these bioactive peptides, but many of these methods suffer from a lack of orthogonality between pairs of protected cysteine (Cys) residues, efficiency, and high yields. Here, we show the utilization of 2,2′‐dipyridyl diselenide (PySeSePy) as a chemical tool for the removal of Cys‐protecting groups and regioselective formation of disulfide bonds in peptides. We found that peptides containing either Cys(Mob) or Cys(Acm) groups treated with PySeSePy in trifluoroacetic acid (TFA) (with or without triisopropylsilane (TIS) were converted to Cys‐S–SePy adducts at 37 °C and various incubation times. This novel Cys‐S–SePy adduct is able to be chemoselectively reduced by five‐fold excess ascorbate at pH 4.5, a condition that should spare already installed peptide disulfide bonds from reduction. This chemoselective reduction by ascorbate will undoubtedly find utility in numerous biotechnological applications. We applied our new chemistry to the iodine‐free synthesis of the human intestinal hormone guanylin, which contains two disulfide bonds. While we originally envisioned using ascorbate to chemoselectively reduce one of the formed Cys‐S–SePy adducts to catalyze disulfide bond formation, we found that when pairs of Cys(Acm) residues were treated with PySeSePy in TFA, the second disulfide bond formed spontaneously. Spontaneous formation of the second disulfide is most likely driven by the formation of the thermodynamically favored diselenide (PySeSePy) from the two Cys‐S–SePy adducts. Thus, we have developed a one‐pot method for concomitant deprotection and disulfide bond formation of Cys(Acm) pairs in the presence of an existing disulfide bond.     

Assignment of the disulfide bonds in the sweet protein brazzein

The thermostable sweet protein brazzein consists of 54 amino acid residues and has four intramolecular disulfide bonds, the location of which is unknown. We found that brazzein resists enzymatic hydrolysis at enzyme/substrate ratios (w/w) of 1:100-1:10 at 35–40°C for 24–48 h. Brazzein was hydrolyzed using thermolysin at an enzyme/substrate ratio of 1:1 (w/w) in water, pH 5.5. for 6 h and at 50°C. The disulfide bonds were determined, by a combination of mass spectrometric analysis and amino acid sequencing of cystine-containing peptides, to be between Cys4-Cys52, Cys16-Cys37, Cys22-Cys47, and Cys26-Cys49. These disulfide bonds contribute to its thermostability. © 1996 John Wiley & Sons, Inc.