Determination of disulfide bond arrangement in bombyxin-IV,an insulin superfamily peptide from the silkworm,Bombyx mori,by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers

The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6–A11, A7–B10, and A20–B22. An intermolecular bond of A20–B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6–A11, A7–B10, A20–B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6–A7, A11–B10, A20–B22-cystine]- and [A6–B10, A7–A11, A20–B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.     

Synthesis of bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by stepwise and selective formation of three disulfide bridges.

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm,Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for -amino group. Three disulfide bonds were bridged step by step (A6–A11, A20–B22, and A7–B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.     

Synthesis of bombyxin-IV,an insulin superfamily peptide from the silkworm,Bombyx mori,by stepwise and selective formation of three disulfide Bridges

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm,Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for α-amino group. Three disulfide bonds were bridged step by step (A6–A11, A20–B22, and A7–B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.     

Synthesis of bombyxin-IV, an insulin-like heterodimeric peptide from the silkworm, Bombyx mori

Bombyxin-IV, a molecular species of bombyxin, which is a member of insulin-like heterodimeric peptides of the silkworm Bombyx mori with prothoracicotropic hormone activity, was synthesized. The A- and B-chains of bombyxin-IV containing four and two Cys residues, respectively, were first synthesized separately by solid phase chemistry using Boc protocol. Then they were coupled by stepwise removal of two different protecting groups at the cysteinyl thiols for semiselective formation of disulfide bridges to give bombyxin-IV in 8% yield. The synthetic bombyxin-IV was shown to have chromatographic and biological properties identical with those of natural bombyxin-IV.     

Bombyxin-II and its disulfide bond isomers: synthesis and activity.

Bombyxin-II, an insulin superfamily peptide of the silkmoth Bombyx mori, and its disulfide bond isomers have been synthesized by two ways of stepwise, semi-regioselective disulfide bond formation. The disulfide bond CysA20-CysB22 or CysA7-CysB10 was formed first, and then the two other disulfide bonds were formed by iodine oxidation. The conditions for the iodine oxidation were improved to suppress oxidative degradation of unprotected Trp residues. With these conditions, bombyxin-II was synthesized in high yields (26% and 32%). Its disulfide bond isomers were also obtained. Specific activity of the products indicates that the disulfide bond CysA20-CysB22 is important to the bombyxin activity.     

A protein caught in a kinetic trap: structures and stabilities of insulin disulfide isomers

Proinsulin contains six cysteines whose specific pairing (A6-A11, A7-B7, and A20-B19) is a defining feature of the insulin fold. Pairing information is contained within A and B domains as demonstrated by studies of insulin chain recombination. Two insulin isomers containing non-native disulfide bridges ([A7-A11,A6-B7,A20-B19] and [A6-A7,A11-B7,A20-B19]), previously prepared by directed chemical synthesis, are metastable and biologically active. Remarkably, the same two isomers are preferentially formed from native insulin or proinsulin following disulfide reassortment in guanidine hydrochloride. The absence of other disulfide isomers suggests that the observed species exhibit greater relative stability and/or kinetic accessibility. The structure of the first isomer ([A7-A11,A6-B7,A20-B19], insulin-swap) has been described [Hua, Q. X., Gozani, S. N., Chance, R. E., Hoffmann, J. A., Frank, B. H., and Weiss, M. A. (1995) Nat. Struct. Biol. 2, 129-138]. Here, we demonstrate that the second isomer (insulin-swap2) is less ordered than the first. Nativelike elements of structure are retained in the B chain, whereas the A chain is largely disordered. Thermodynamic studies of guanidine denaturation demonstrate the instability of the isomers relative to native insulin (DeltaDeltaG(u) > 3 kcal/mol). In contrast, insulin-like growth factor I (IGF-I) and the corresponding isomer IGF-swap, formed as alternative products of a bifurcating folding pathway, exhibit similar cooperative unfolding transitions. The insulin isomers are similar in structure and stability to two-disulfide analogues whose partial folds provide models of oxidative folding intermediates. Each exhibits a nativelike B chain and less-ordered A chain. This general asymmetry is consistent with a hierarchical disulfide pathway in which nascent structure in the B chain provides a template for folding of the A chain. Structures of metastable disulfide isomers provide probes of the topography of an energy landscape.     

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.     

Effect of reduction of the interchain disulfide bridge of human thrombin on its enzymatic activity and structure

It was shown that selective hydrolysis of the disulfide bridge between the A- and B-chains of human thrombin in the absence of denaturating agents decrease its proteolytic (e.g., fibrinogen-binding), esterase and amidase activities. Both chains remain bound by non-covalent interactions. A preparation of partially reduced thrombin was obtained and its kinetic parameters were determined. The experimental results suggest that the S-S bond connecting the A- and B-chains of thrombin is involved in the stabilization of the enzyme active center.     

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

Role of disulfide bonds in the structure and activity of human insulin

Insulin contains two inter-chain disulfide bonds between the A and B chains (A7-B7 and A20-B19), and one intra-chain linkage in the A chain (A6-A11). To investigate the role of each disulfide bond in the structure, function and stability of the molecule, three des mutants of human insulin, each lacking one of the three disulfide bonds, were prepared by enzymatic conversion of refolded mini-proinsulins. Structural and biological studies of the three des mutants revealed that all three disulfide bonds are essential for the receptor binding activity of insulin, whereas the different disulfide bonds make different contributions to the overall structure of insulin. Deletion of the A20-B19 disulfide bond had the most substantial influence on the structure as indicated by loss of ordered secondary structure, increased susceptibility to proteolysis, and markedly reduced compactness. Deletion of the A6-A11 disulfide bond caused the least perturbation to the structure. In addition, different refolding efficiencies between the three des mutants suggest that the disulfide bonds are formed sequentially in the order A20-B19, A7-B7 and A6-A11 in the folding pathway of proinsulin.     

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

Opsin stability and folding: the role of Cys185 and abnormal disulfide bond formation in the intradiscal domain

The structure in the extracellular, intradiscal domain of rhodopsin surrounding the Cys110–Cys187 disulfide bond has been shown to be important for correct folding of this receptor in vivo. Retinitis pigmentosa misfolding mutants of the apoprotein opsin (such as P23H) misfold, as defined by a deficiency in ability to bind 11-cis retinal and form rhodopsin. These mutants also possess an abnormal Cys185–Cys187 disulfide bond in the intradiscal domain. Here, by mutating Cys185 to alanine, we eliminate the possibility of forming this abnormal disulfide bond and investigate the effect of combining the C185A mutation with the retinitis pigmentosa mutation P23H. Both the P23H and P23H/C185A double mutant suffer from low expression and poor 11-cis retinal binding. Our data suggest that misfolding events occur that do not have an absolute requirement for abnormal Cys185–Cys187 disulfide bond formation. In the detergent-solubilised, purified state, the C185A mutation allows formation of rhodopsin at wild-type (WT) levels, but has interesting effects on protein stability. C185A rhodopsin is less thermally stable than WT, whereas C185A opsin shows the same ability to regenerate rhodopsin in detergent as WT. Purified C185A and WT opsins, however, have contrasting 11-cis retinal binding kinetics. A high proportion of C185A opsin binds 11-cis retinal with a slow rate that reflects a denatured state of opsin reverting to a fast-binding, open-pocket conformation. This slower rate is not observed in a stabilising lipid/detergent system, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Chaps, in which C185A exhibits WT (fast) retinal binding. We propose that the C185A mutation destabilises the open-pocket conformation of opsin in detergent resulting in an equilibrium between correctly folded and denatured states of the protein. This equilibrium can be driven towards the correctly folded rhodopsin state by the binding of 11-cis retinal.     

Heat treatment of bovine alpha-lactalbumin results in partially folded, disulfide bond shuffled states with enhanced surface activity

Prolonged heating of holo bovine alpha-lactalbumin (BLA) at 80 degrees C in pH 7 phosphate buffer in the absence of a thiol initiator improves the surface activity of the protein at the air:water interface, as determined by surface tension measurements. Samples after 30, 60, and 120 min of heating were analyzed on cooling to room temperature. Size-exclusion chromatography shows sample heterogeneity that increases with the length of heating. After 120 min of heating monomeric, dimeric, and oligomeric forms of BLA are present, with aggregates formed from disulfide bond linked hydrolyzed protein fragments. NMR characterization at pH 7 in the presence of Ca2+ of the monomer species isolated from the sample heated for 120 min showed that it consisted of a mixture of refolded native protein and partially folded protein and that the partially folded protein species had spectral characteristics similar to those of the pH 2 molten globule state of the protein. Circular dichroism spectroscopy showed that the non-native species had approximately 40% of the alpha-helical content of the native state, but lacked persistent tertiary interactions. Proteomic analysis using thermolysin digestion of three predominant non-native monomeric forms isolated by high-pressure liquid chromatography indicated the presence of disulfide shuffled isomers, containing the non-native 61-73 disulfide bond. These partially folded, disulfide shuffled species are largely responsible for the pronounced improvement in surface activity of the protein on heating.     

Determination of the disulfide bond pairings in bovine transforming growth factor-alpha

A rapid method for determining the three disulfide bond pairings in bovine transforming growth factor-alpha (bTGF-alpha) was developed by digesting bTGF-alpha with thermolysin followed by separation of the generated peptides by reversed-phase HPLC. The disulfide-bonded peptides were identified by amino acid sequencing and fast atom bombardment mass spectrometry. The disulfide bond pairings in bTGF-alpha were determined to be homologous to those in the human and mouse TGF-alpha molecules. A species of low bioactivity isolated from the folding/oxidation mixture of chemically synthesized bTGF-alpha was demonstrated to contain two incorrect disulfide bonds. These results indicate that mispairing of disulfide bonds in bTGF-alpha significantly reduces the activity of this molecule.     

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.     

Structure-activity relationships of the intramolecular disulfide bonds in coprisin,a defensin from the dung beetle

Defensins, which are small cationic molecules produced by organisms as part of their innate immune response, share a common structural scaffold that is stabilized by three disulfide bridges. Coprisin is a 43-amino acid defensin-like peptide from Copris tripartitus. Here, we report the intramolecular disulfide connectivity of cysteine-rich coprisin, and show that it is the same as in other insect defensins. The disulfide bond pairings of coprisin were determined by combining the enzymatic cleavage and mass analysis. We found that the loss of any single disulfide bond in coprisin eliminated all antibacterial, but not antifungal, activity. Circular dichroism (CD) analysis showed that two disulfide bonds, Cys20-Cys39 and Cys24-Cys41, stabilize coprisin’s α-helical region. Moreover, a BLAST search against UniProtKB database revealed that coprisin’s α-helical region is highly homologous to those of other insect defensins. [BMB Reports 2014; 47(11): 625-630]     

Effects of deletion and shift of the A20-B19 disulfide bond on the structure, activity, and refolding of proinsulin

To investigate the role of the A20-B19 disulfide bond in the structure, activity and folding of proinsulin, a human proinsulin (HPI) mutant [A20, B19Ala]-HPI was prepared. This mutant, together with another proinsulin mutant previously constructed with an A19Tyr deletion, which can also be taken as shifted mutant of the A20-B19 disulfide bond, were studied for their in vitro refolding, oxidation of free thiol groups, circular dichroism spectra, antibody and receptor binding activities and sensitivity to trypsin digestion in comparison with native proinsulin. The results indicate that deletion of the A20-B19 disulfide bond results in a large decrease in the alpha-helix content of the molecule and higher sensitivity to tryptic digestion. Both the deletion and shift mutations, especially the latter, cause a great decrease in the biological activity of proinsulin analogues. The folding yields of HPI analogues were much lower than that of HPI. And the shift mutant, [Delta A19Tyr]-HPI, was scarcely refolded correctly in vitro and its refolding yield was extremely low. These results suggest that the A20-B19 disulfide bond plays an important role in the structural stabilization and folding of the insulin precursor. By summarizing the refolding studies on proinsulin, a possible folding pathway is proposed.     

Stereoselective disulfide formation stabilizes the local peptide conformation in nisin mimics

Nisin is a polymacrocyclic peptide antimicrobial with high activity against Gram-positive bacteria. Lanthionine and methyllanthionine bridges, closing the macrocycles, are stabilized by thioether bonds, formed between cysteines and dehydrated serine or threonine. The role of polypeptide backbone conformation in the formation of macrocycles A and B within cysteine mutants of nisin residues 1?12 is investigated here by molecular dynamics simulations. Enantiomeric combinational space of Cys3 and Cys7 and of Cys8 and Cys11 is examined for the preference of disulfide bond formation over helical turn formation within this region. A clear preference for spontaneous disulfide formation and closure of rings 3,7 and 8,11 is demonstrated for the D-Cys3, D-Cys7, L-Cys8, L-Cys11 nisin homologue, while interlinked rings A and B are obtained through disulfide bridges between L-Cys3 and D-Cys8 and between D-Cys7 and D-Cys11. This study offers a simple designer approach to solid phase synthesis of macrocyclic peptides and lantibiotic analogues.     

Striking stabilization of Arc repressor by an engineered disulfide bond

A solvent-exposed Cys11-Cys11' disulfide bond was designed to link the antiparallel strands of the beta sheet both in the Arc repressor dimer and in a single-chain variant in which the Arc subunits are connected by a 15-residue peptide tether. In both proteins, the presence of the disulfide bond increased the T(m) by approximately 40 degrees C. In the single-chain background, the disulfide bond stabilized Arc by 8.5 kcal/mol relative to the reduced form, a significantly larger degree of stabilization than caused by other engineered disulfides and most natural disulfides. This exceptional stabilization arises from a modest effective concentration of the Cys11-Cys11' disulfide in the native state (71 M) and an anomalously low effective concentration in the denatured state (40 microM). Disulfide cross-linking of the two beta strands in the single-chain Arc background accelerated refolding by a factor of 170 into the sub-microsecond time scale. However, the major energetic effect of the disulfide occurs after the transition state for Arc refolding, slowing unfolding by 200 000-fold.     

Complete structure of eclosion hormone of Manduca sexta. Assignment of disulfide bond location.

The locations of the three disulfide bonds of eclosion hormone (EH) isolated from Manduca sexta were assigned by sequence analysis of thermolysin fragments and by comparison of a key heterodimeric fragment to regiospecifically synthesized parallel and antiparallel isomers. We elucidated the complete structure of Manduca EH as a 62-residue peptide which has three disulfide bonds between Cys14-Cys38, Cys18-Cys34, and Cys21-Cys49.