The folding nucleus of the insulin superfamily: a flexible peptide model foreshadows the native state

Oxidative folding of insulin-like growth factor I (IGF-I) and single-chain insulin analogs proceeds via one- and two-disulfide intermediates. A predominant one-disulfide intermediate in each case contains the canonical A20-B19 disulfide bridge (cystines 18-61 in IGF-I and 19-85 in human proinsulin). Here, we describe a disulfide-linked peptide model of this on-pathway intermediate. One peptide fragment (19 amino acids) spans IGF-I residues 7-25 (canonical positions B8-B26 in the insulin superfamily); the other (18 amino acids) spans IGF-I residues 53-70 (positions A12-A21 and D1-D8). Containing only half of the IGF-I sequence, the disulfide-linked polypeptide (designated IGF-p) is not well ordered. Nascent helical elements corresponding to native alpha-helices are nonetheless observed at 4 degrees C. Furthermore, (13)C-edited nuclear Overhauser effects establish transient formation of a native-like partial core; no non-native nuclear Overhauser effects are observed. Together, these observations suggest that early events in the folding of insulin-related polypeptides are nucleated by a native-like molten subdomain containing Cys(A20) and Cys(B19). We propose that nascent interactions within this subdomain orient the A20 and B19 thiolates for disulfide bond formation and stabilize the one-disulfide intermediate once formed. Substitutions in the corresponding region of insulin are associated with inefficient chain combination and impaired biosynthetic expression. The intrinsic conformational propensities of a flexible disulfide-linked peptide thus define a folding nucleus, foreshadowing the structure of the native state.     

Identification of insulin intermediates and sites of cleavage of native insulin by insulin protease from human fibroblasts

We have studied the time sequence degradation of native insulin by insulin protease from human fibroblast using multiple steps involving purification of the products by high performance liquid chromatography, determination of peak composition by amino acid sequence analysis, and confirmation of structure by mass spectrometry and thus elucidated the sites of cleavage of insulin by human insulin protease. We observed that as early as 0.5 min of incubation, three major new peptide peaks, intact insulin, and four smaller peptide peaks can be detected. The major peptides are portions of the insulin molecule, with the amino ends of the A and B chains or the carboxyl ends of the A and B chains still connected by disulfide bonds. Peptide peak I is A1-13-B1-9. Peptide peak II is A1-14-B1-9. Peptide peak III is A14-21-B14-30. The smaller peptide peaks are A14-21-B17-30, A15-21-B14-30, A15-21-B10-30, and A14-21-B10-30. The major peptide bond cleavage sites therefore consist of A13-14, A14-15, B9-10, B13-14, and B10-17. With longer incubation times, peptide peak II appears to lose the A14 tyrosine to form peptide peak I. This peptide I, which is the amino end of the A and B chains, is not further degraded even after 1.5 h of incubation. With longer incubation times, the peptides containing the carboxyl ends of the A and B chains are further degraded to form products from cleavage at the A18-19, B14-15, B25-26, and a small amount of A19-20, B10-11, and B24-25 cleavage and the emergence of 2-5-amino acid peptide chains, tyrosine, alanine, histidine, and leucine-tyrosine. We conclude, based on the three-dimensional structure of insulin, that human insulin protease recognizes the alpha-helical regions around leucine-tyrosine bonds and that final degradation steps to small peptides do not require lysosomal involvement.     

Synthesis of cystine peptides 21-25/70-73 and 35-39/56-59 of the beta-subunit of human choriogonadotropin.

Syntheses of two asymmetrical cystine peptides with the amino acid residues 21-25/70-73 and 35-39/56-59, based on the linear amino acid sequence and the disulfide bond assignment in the beta-subunit of human choriogonadotropin (hCG-beta), are described. S-trityl and S-acetamidomethyl peptide fragments of each cystine peptide were prepared in solution phase and were subjected to oxidation with I2/MeOH to form the disulfide bridge. The cystine peptides were characterized by their amino acid analyses and fast atom bombardment mass spectrometry. Immunological characterization by several homologous radioimmunoassay systems showed that peptide 21-25/70-73 had significant hCG, hCG-beta, and hLH activities while peptide 35-39/56-59 failed to reveal any immunoreactivity.     

Synthetic insulin fragment with insulin-like biological activity.

An insulin fragment, representing the C-terminal functionally important site of its molecule and responsible for receptor binding, was synthesized. The fragment consists of two peptides: a dipeptide (A 20-21) and an octapeptide (B 19-26), linked with a disulfide bond (A20-B19). The biological activity of the newly synthesized fragment relative to insulin was assayed for the influence on glycogenesis and for the ability to stimulate glucose uptake. Comparative tests for the biological activity of the synthesized fragment and of the intact hormone allowed us to conclude that the fragment has insulin-like properties.     

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.     

Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide curvacin A

The 3D structure of the membrane-permeabilizing 41-mer pediocin-like antimicrobial peptide curvacin A produced by lactic acid bacteria has been studied by NMR spectroscopy. In DPC micelles, the cationic and hydrophilic N-terminal half of the peptide forms an S-shaped beta-sheet-like domain stabilized by a disulfide bridge and a few hydrogen bonds. This domain is followed by two alpha-helices: a hydrophilic 6-mer helix between residues 19 and 24 and an amphiphilic/hydrophobic 11-mer helix between residues 29 and 39. There are two hinges in the peptide, one at residues 16-18 between the N-terminal S-shaped beta-sheet-like structure and the central 6-mer helix and one at residues 26-28 between the central helix and the 11-mer C-terminal helix. The latter helix is the only amphiphilic/hydrophobic part of the peptide and is thus presumably the part that penetrates into the hydrophobic phase of target-cell membranes. The hinge between the two helices may introduce the flexibility that allows the helix to dip into membranes. The helix-hinge-helix structure in the C-terminal half of curvacin A clearly distinguishes this peptide from the other pediocin-like peptides whose structures have been analyzed and suggests that curvacin A along with the structural homologues enterocin P and carnobacteriocin BM1 belong to a subgroup of the pediocin-like family of antimicrobial peptides.     

Selective proteolytic cleavage of recombinant human interleukin 4. Evidence for a critical role of the C-terminus

Human interleukin 4 is a 129 amino acid lymphokine secreted by activated T cells that exerts pleiotropic biological effects on B and T lymphocytes and other hematopoietic cells. Structure-function relations were studied by employing selective proteolytic cleavage of purified recombinant human interleukin 4 (rhuIL-4). Limited proteolysis with endoprotease Glu-C from Staphylococcus aureus (V8) produced two digestion products that were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 19K (I) and 15K (II), respectively. These species were isolated by reversed-phase HPLC. Amino acid sequencing indicated that species II was an 84 amino acid core fragment extending from Gln-20 to Glu-103 and containing a hydrolyzed peptide bond at Glu-26. On the basis of known disulfide bond assignments, it was concluded that species II was stabilized by two disulfide bonds (Cys-24/Cys-65 and Cys-46/Cys-99). Analysis of its secondary structure by circular dichroism revealed a high content of alpha helix. Species I was the full-length rhuIL-4 with selective cleavage at Glu-26 and Glu-103. Both species I and II were inactive in an in vitro assay based on proliferation of peripheral blood lymphocyte blasts and lacked the ability to bind to teh rhuIL-4 receptor on Daudi cells. In order to elucidate further the role of the residues removed by S. aureus V8 protease, rabbit antisera were raised to synthetic peptides corresponding to residues 1-26 at the N-terminus and 104-129 at the C-terminus. Only antisera directed to the C-terminal peptide inhibited binding of 125I-rhuIL-4 to Daudi cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis

A cDNA clone, pFL18, carrying a putative full-length fibroin light chain (L-chain) sequence was isolated and its nucleotide sequence was determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 262 amino acid residues. The sequence was concluded to be that of the L-chain with its signal peptide because corresponding amino acid sequences for the seven tryptic and the four chymotryptic peptides from the purified L-chain were all included and an N-terminal region having typical properties of a signal peptide was present. The N terminus of the mature form of L-chain was identified as N-acetyl serine by analyzing the acyl-dansylhydrazide derived from the N-acyl-amino acid which had been released from the N-terminal blocked chymotryptic peptide by the acylamino acid-releasing enzyme. It was suggested that a signal peptide had cleaved between Pro18 and Ser19, yielding a mature L-chain polypeptide consisting of 244 amino acid residues. The molecular weight of the L-chain was calculated to be 25,800 including the N-acetyl group. The L-chain contained three Cys residues, two of which were suggested to form an intramolecular disulfide linkage, leaving the third one at the most C-terminal position and in a relatively hydrophilic region as the most probable site of disulfide linkage with the fibroin heavy chain.     

胰岛素折叠、结合与稳定性的核磁共振研究(英)

Insulin is one of the most important hormonal regulators of metabolism. Since the diabetes patients increase dramatically, the chemical properties, biological and physiological effects of insulin had been extensively studied. In last decade the development of NMR technique allowed us to determine the solution structures of insulin and its variety mutants in various conditions, so that the knowledge of folding, binding and stability of insulin in solution have been largely increased. The solution structure of insulin monomers is essentially identical to those of insulin monomers within the dimer and bexamer as determined by X-ray diffraction. The studies of insulin mutants at the putative residues for receptor binding explored the possible conformational change and fitting between insulin and its receptor. The systematical studies of disulfide paring coupled insulin folding intermediates revealed that in spite of the conformational variety of the intermediates, one structural feature is always remained: a “native-like B chain super-secondary structure“, which consists of B9-B19 helix with adjoining B23-B26 segment folded back against the central segment of B chain, an internal cystine A20-B19 disulfide bridge and a short a-helix at C-terminal of A chain linked. The “super-secondary structure“ might be the “folding nucleus“ in insulin folding mechanism. Cystine A20-B19 is the most important one among three disulfides to stabilize the nascent polypeptide in early stage of the folding. The NMR structure of C. elegans insulin-like peptide resembles that of human insulin and the peptide interacts with human insulin receptor. Other members of insulin superfamily adopt the “insulin fold“ mostly. The structural study of insulin-insulin receptor complex, that of C elegans and other invertebrate insulin-like peptide, insulin fibril study and protein disulfide isomerase (PDI) assistant proinsulin folding study will be new topics in future to get insight into folding, binding, stability, evolution and fibrillation of insulin in detail.     

Conformation of the 18-23 loop region of bovine prothrombin in the absence and presence of a model Ca2+ ion. An energy minimization study

The conformation of the cyclic peptide Ac-Cys-Leu-Gla-Gla-Pro-Cys-NHMe, representing the 18-23 disulfide loop of bovine prothrombin, was studied by energy minimization with the ECEPP (Empirical Conformational Energy Program for Peptides) algorithm. Parameters for charge and geometry for the gamma-carboxyglutamic acid (Gla) residue were obtained for inclusion in the ECEPP data set. Construction of the 18-23 cyclic peptide, for which no crystal structure is available, was carried out by using a scheme that took advantage of the constraints imposed by the requirement of disulfide ring closure and utilized known low-energy structures of single residues and dipeptides. Both cis and trans isomers about the Gla 21-Pro 22 peptide bond were considered. The lowest-energy conformation found for the isolated 18-23 cyclic peptide with arbitrary reduction of the charge on the Gla residues (to simulate hydration roughly) is a trans form, differing in energy by 11 kcal-mol-1 from the lowest-energy cis form. However, when the energy calculation includes one model Ca2+ ion, X2+, introduced at a fixed distance of 2.3 A from a single oxygen atom of either of the side-chain carboxyl groups of Gla with the C delta-O-X2+ bond angle fixed at one of three values, the lowest-energy cis conformation is about 1 kcal-mol-1 lower in energy than the lowest-energy trans conformation; i.e. the two structures have similar energies. In these structures, four oxygen atoms, two from each Gla side-chain, approach the model Ca2+ ion closely, in a manner similar to that seen in crystals of calcium alpha-ethylmalonate (Zell, A., Einspahr, H. & Bugg, C.E. (1985) Biochemistry 24, 533-537). It appears that the binding of Ca2+ to the 18-23 cyclic peptide may alter the equilibrium between cis and trans structures such that the fraction of cis isomers is greater in the presence of Ca2+.     

A peptide model of insulin folding intermediate with one disulfide

Insulin folds into a unique three-dimensional structure stabilized by three disulfide bonds. Our previous work suggested that during in vitro refolding of a recombinant single-chain insulin (PIP) there exists a critical folding intermediate containing the single disulfide A20-B19. However, the intermediate cannot be trapped during refolding because once this disulfide is formed, the remaining folding process is very quick. To circumvent this difficulty, a model peptide ([A20-B19]PIP) containing the single disulfide A20-B19 was prepared by protein engineering. The model peptide can be secreted from transformed yeast cells, but its secretion yield decreases 2-3 magnitudes compared with that of the wild-type PIP. The physicochemical property analysis suggested that the model peptide adopts a partially folded conformation. In vitro, the fully reduced model peptide can quickly and efficiently form the disulfide A20-B19, which suggested that formation of the disulfide A20-B19 is kinetically preferred. In redox buffer, the model peptide is reduced gradually as the reduction potential is increased, while the disulfides of the wild-type PIP are reduced in a cooperative manner. By analysis of the model peptide, it is possible to deduce the properties of the critical folding intermediate with the single disulfide A20-B19.     

Determination of the spatial structure of insectotoxin 15A from Buthus erpeus by (1)H-NMR spectroscopy data]

The solution structure of insectotoxin 15A (35 residues) from scorpion Buthus eupeus was determined on the basis of 386 interproton distance restraints 12 hydrogen-bonding restraints and 113 dihedral angle restraints derived from 1H NMR experiments. A group of 20 structures was calculated with the distance geometry program DIANA followed by the restrained energy minimization with the program CHARMM. The atomic RMS distribution about the mean coordinate position is 0.64 +/- 0.11 A for the backbone atoms and 1.35 +/- 0.20 A for all atoms. The structure contains an alpha-helix (residues 10-20) and a three-stranded antiparallel beta-sheet (residues 2-5, 24-28 and 29-33). A pairing of the eight cysteine residues of insectotoxin 15A was established basing on NMR data. Three disulfide bridges (residues 2-19, 16-31 and 20-33) connect the alpha-helix with the beta-sheet, and the fourth one (5-26) joins beta-strands together. The spatial fold of secondary structure elements (the alpha-helix and the beta-sheet) of the insectotoxin 15A is very similar to those of the other short and long scorpion toxins in spite of a low (about 20%) sequence homology.     

New potent hGH-RH analogues with increased resistance to enzymatic degradation.

Four hGH-RH analogues containing homoarginine (Har) and/or D-Arg were obtained by solid-phase methodology using Boc-chemistry. To introduce Har residues, a Lys(Fmoc) protected Lys derivative was incorporated in the appropriate positions (11, 12, 20, 21 or 29): after assembly of the peptide chain the Fmoc group was removed and the peptide-resin was guanidinylated by treatment with N,N'-bis(tert-butoxycarbonyl)-S-methylisothiourea. The peptides were cleaved from the resin by treatment with liquid HF, and the products were purified by RP-HPLC. The peptides were subjected to digestion by trypsin, and the course of the reaction was followed by HPLC and ESI-MS. It was found that peptide bonds formed by the carboxyl group of Har are completely stable to trypsin. The course of cleavage at Lys or Arg residues depends on the position of Har in the sequence. All the analogues investigated stimulate the release of GH in rats after subcutaneous administration, and were about 50-100 times as potent as rGH-RH itself. The analogues had no effect on PRL, LH and FSH levels.     

Characterization and cDNA cloning of androgenic gland hormone of the terrestrial isopod Armadillidium vulgare.

The sex differentiation in crustaceans is known to be controlled by a peptide hormone called androgenic gland hormone (AGH). AGH was extracted and purified from the androgenic glands (AGs) of the male isopod Armadillidium vulgare by high-performance liquid chromatography. AGH consisted of two peptide chains and their N-terminal amino acid sequences were determined. A cDNA encoding AGH was cloned by PCR and sequenced. The cDNA had an open reading frame of 432 bp, which encoded a preproAGH consisting of a signal peptide (21 residues), B chain (44 residues), C peptide (46 residues), and A chain (29 residues). Through processing, the A and B chains might form a heterodimer interlinked by disulfide bonds. The A chain possessed a putative N-linked glycosylation site. A Northern blot analysis using the cDNA as a probe detected a hybridization signal with 0.8 kb in the RNA preparation only from the AGs.     

Primary structure of potato kunitz-type serine proteinase inhibitor

The serine proteinase inhibitor (PSPI-21) isolated from potato tubers (Solanum tuberosum L.) comprises two protein species with pI 5.2 and 6.3, denoted as PSPI-21-5.2 and PSPI-21-6.3, respectively. They were separated by anion exchange chromatography on a Mono Q FPLC column. Both species tightly inhibit human leukocyte elastase, whereas their interaction with trypsin and chymotrypsin is substantially weaker. The sequences of both PSPI-21-5.2 and PSPI-21-6.3 were determined by analysis of overlapping peptides obtained from the oxidized or reduced and S-pyridylethylated proteins after digestion with trypsin or pepsin. Both species of PSPI-21 are composed of two chains, named chains A and B, which are linked by a disulfide bridge between Cys(146) and Cys(157). The other disulfide bridge is located within the A chains between Cys(48) and Cys(97). The amino acid sequences of the large A chains of the two forms, consisting of 150 amino acids residues each, differ in a single residue at position 52. The small chains B, containing 37 and 36 residues in PSPI-21-6.3 and PSPI-21-5.2, respectively, have nine different residues. The entire amino acid sequences of the two inhibitors show a high degree of homology to the other Kunitz-type proteinase inhibitors from plants.     

Synthesis of the trypsin fragment 10-25/75-88 of mouse nerve growth factor. I. The protected tetradecapeptide 75-88

Cyanogen bromide cleavage followed by trypsin digestion of mouse nerve growth factor (NGF) allowed the isolation of a double chain unsymmetrical cystine peptide of high neurotrophic activity. The presence of tryptophan residues severely limits the synthetic approaches for selective disulfide bridging. The strategy applied for such a purpose as well as the preparation of the suitably protected tetradecapeptide corresponding to sequence 75-88 as key intermediate for the synthesis of the NGF fragment 10-25/75-88 are described.     

Use of local electrostatic environments of cysteines to enhance formation of a desired species in a reversible disulfide exchange reaction

The role of electrostatic factors has been evaluated for the reversible disulfide exchange reaction between N-acetylcysteine (A) and a peptide fragment (B) comprising residues 85-114 of Kunitz soybean trypsin inhibitor. In A, the sulfhydryl group has a negative carboxyl neighbor on the cysteine itself. In B, the only charged group within five residues of the single cysteine at position 86 is the positive N-terminal amino group on residue 85. The concentrations of the monomers A and B and of the disulfides AA, AB and BB have been determined as a function of time in kinetic experiments at pH 7, 23 degrees C and ionic strengths of 20 mM and 1 M. At both ionic strengths the sulfhydryl acid dissociation constants Ka have been determined for A and B, as well as the four rate constants for the disulfide exchange reaction. The electrostatic effects are small in magnitude but occur in expected directions. Local cysteine environments enhance formation of the mixed disulfide (AB), having a favorable configuration of adjacent unlike charges and generate decreases in the AA and BB disulfides joining regions of identical charge. These experiments represent an initial step towards use of intrinsic protein functional groups to direct formation of specific disulfides in a synthetic protein.     

Solid phase synthesis and biological activity of mast cell degranulating (MCD) peptide: a component of bee venom

Mast cell degranulating (MCD) peptide, a 22 amino acid residue basic peptide from bee venom, was synthesized by stepwise solid phase synthesis on a benzhydrylamine resin support. N alpha-t-butyloxycarbonyl and benzyl type side chain protection was used. The two disulfide bridges were formed selectively by using S-acetamidomethyl protection for the cysteine residues in position 5 and 19 and S-methylbenzyl protection for the cysteine residues in positions 3 and 15. Crude synthetic MCD peptide was obtained following deprotection and cleavage from the resin by the low/high HF method. The peptide was isolated in pure form by ion exchange chromatography and gel filtration. The final product has physical, chemical, and biological properties identical with those reported for the natural product. The synthetic strategy utilized for MCD peptide will facilitate the availability of structurally similar analogs for evaluating antihistaminic and anti-inflammatory activities.     

Solid-phase synthesis of huwentoxin-I and its structure and bioactivity analysis

Huwentoxin-I, a neurotoxic peptide from the spider Selenocosmia huwena, was synthesized by sol-id-phase method with Fluorenylmethoxycarbonyl amino acid pentafluorophenyl esters (Fmoc-AA-OPfp). The carboxyl and the hydroxy groups were protected by tBu; the side chains of Lys and His were protected by Roc; the guanidine group of Arg was protected by Mtr and the mercaptan group of Cys was protected by Trt. The solid-phase carrier was ethylene diamine-polyethylene-polystyrene (DEA-PEG-PS) resin. The synthetic peptide was cleaved from the resin and deprotected by a 90% TFA solution containing 5% thioanisole, 3% ethanedithiol and 2% anisole. The product was reduced with DTT and then incubated with GSSG and GSH to form the correct disulfide bond linkages. The syn-thetic peptide was purified by HPLC and then characterized by amino acid composition and sequence analysis, peptide mapping and NMR. The biological activity of the synthetic product was tested by electrophysiological method using the isolated mouse ph     

Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding

We have studied the sensitivity of sulfhydryl groups of a highly purified p21 protein of the v-rasH oncogene to a thiol-specific reagent, N-ethylmaleimide (NEM). Approximately 70% of GTP binding and autokinase activities of p21 were inactivated by NEM, and excessive amounts of GTP or GDP protected p21 activities. Thiol titration revealed the presence of one fast reactive cysteine residue, the susceptibility of which is modulated by GTP binding. A total of 4 and 6 residues, respectively, became titratable upon denaturation and reduction, suggesting the presence of a disulfide bond. This GTP-modulated sulfhydryl group was identified as Cys-80 in the following tryptic peptide sequence: NH2-Thr-Gly-Glu-Gly-Phe-Leu-Cys-Val-Phe-Ala-Ile-Asn-Asn-Thr-Lys-COOH. This is based on the comparative tryptic peptide mapping of [14C]NEM-modified p21 in the presence and absence of GTP. The GTP-modulated peptide co-chromatographed with a synthetic peptide of the predicted sequence. Amino acid analysis of the purified [14C]NEM-modified peptide from tryptic digests of p21 also confirmed its identity. This region of p21 shares an extensive sequence homology with various G-proteins and appears to be in the vicinity of the GTP-binding domain of these proteins.