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1.
Hirudin variant 1 (HV1), a small protein consisting of 65 amino acids and three disulfide bonds, was synthesized by using Fmoc-based convergent methods on 2-chlorotrityl resin (CLTR). The linear sequence was assembled by the sequential condensation of 7 protected fragments, on the resin-bound 55-65 fragment. The conditions of fragment assembly were carefully studied to determine the most efficient synthetic protocol. Crude reduced [Cys(16, 28)(Acm)]-HV1 thus obtained was easily purified to homogeneity by RP-HPLC. Disulfide bridges were successfully formed by a two-step procedure, involving an oxidative folding step to form Cys(6)-Cys(14) and Cys(22)-Cys(39) linkages, followed by iodine oxidation to form the Cys(16)-Cys(28) bond. The correct disulfide bond alignment was established by peptide mapping using Staphylococcus aureus V8 protease at pH 4.5. 相似文献
2.
Naoyoshi Chino Shigeru Kubo Hideki Nishio Yuji Nishiuchi Masamitsu Nakazato Terutoshi Kimura 《International journal of peptide research and therapeutics》2006,12(3):203-209
Reduced human β-defensin (hBD)-1, -2, -3 and -4 synthesized by Boc chemistry were subjected to oxidative folding reaction under optimal conditions. Among the factors affecting the oxidative folding in the presence of reduced and oxidized glutathione (GSH/GSSG), the buffer concentration and reaction temperature were essential for the predominant formation of the native disulfide structure. The homogeneity of the four synthetic hBDs was confirmed by analytical procedures using RP-HPLC, IEX-HPLC, capillary zone electrophoresis (CZE) and MALDI-TOF MS as well as sequencing, although high temperature (70 °C) was used for the RP-HPLC analysis of hBD-3 and hBD-4 to exclude the influence of equilibrium with the respective conformers having native disulfide pairing. All synthetic hBDs were shown to possess the native disulfide structure by sequential analyses and mass measurements with cystine segments obtained by enzymatic digestion. Upon digestion of hBD-1 and hBD-4 with proline specific endopeptidase, the Cys-X bond was found to be reproducibly cleaved together with the Pro-X bond although the cleavage of Cys-X afforded the appropriate cystine segments for determining the disulfide structure of hBD-1 and hBD-4. With respect to antimicrobial activity against E. coli, the four synthetic hBDs of high homogeneity possessed the same potencies as those reported previously.Australian Peptide Conference Issue 相似文献
3.
J R Charpie E Haber G R Matsueda 《Biochemical and biophysical research communications》1988,152(2):910-915
To mimic the sequence spanning the primary site (the Lys158-Ile159 bond) cleaved by plasmin in its conversion of single-chain urokinase plasminogen activator (scuPA) to urokinase, we synthesized the peptide Cys(Acm)-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile-Gly-Gly-Glu-Phe-Cys [Cys(Acm)scuPA(153-164)Cys]. Immunization of A/J mice with the Cys(Acm)scuPA(153-164)Cys peptide linked to hemocyanin, followed by somatic cell fusion with a myeloma cell line (SP2/0), yielded a monoclonal antibody (SCOOP1) that bound to single-chain urokinase but not to urokinase or plasmin-treated single-chain urokinase. SCOOP1 could discriminate between single-chain urokinase and urokinase by greater than three orders of magnitude. In a radioimmunoassay, Cys(Acm)scuPA(153-164)Cys completely inhibited SCOOP1 binding to single-chain urokinase, whereas an equimolar mixture of two heptapeptides comprising the amino terminal [Cys-scuPA(153-158)] and carboxy terminal [scuPA(159-164)Cys)] halves of the cleavage site peptide did not. Thus the epitope recognized by SCOOP1 includes the Lys158-Ile159 peptide bond. 相似文献
4.
鲑鱼降钙素及其类似物的合成 总被引:1,自引:0,他引:1
鲑鱼降钙素及其类似物的合成潘和平王良友陈正英王芳王会信(军事医学科学院基础医学研究所,北京100850)SynthesisofSalmonCalcitoninandItsAnalogsPanHe-PingWangLiang-YouChenZheng-... 相似文献
5.
Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX. 总被引:1,自引:0,他引:1
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Y. Yang W. V. Sweeney K. Schneider B. T. Chait J. P. Tam 《Protein science : a publication of the Protein Society》1994,3(8):1267-1275
The 45-residue C-terminal EGF-like domain in human blood coagulation factor IX has been synthesized by a 2-step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4-methyl-benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1-3), Cys12 to Cys26 (Cys 2-4), or Cys28 to Cys41 (Cys 5-6). Only the peptide having the Cys 2-4 pair blocked with Acm forms the desired disulfide isomer (Cys 1-3/5-6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF-like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds. 相似文献
6.
Nobutaka Fujii Akira Otaka Susumu Funakoshi Toshihiro Watanabe Hiromitsu Arai Kiyoshi Bessho Haruaki Yajima 《Journal of Protein Chemistry》1988,7(2):151-156
Treatment of a mixture of Cys(R)(O) and Cys(R) with an acid was found to generate cystine in fairly good yields, when suitable R, R, and an acid were selected. An unsymmetrical cystine peptide was prepared by treatment of a mixture of Z(OMe)-Cys(R) (0)-Ala-NH2 (R=Acm or MBzl) and Z(OMe)-Cys(MBzl)-Gly-OBzl with TFA or 1 M TFMSA/TFA.3 Oxytocin was obtained in an excellent yield by TFA treatment of the protected peptide containing Cys(Acm)(0) and Cys(MBzl). Thus, formation of the disulfide bond was found feasible at the position of Cys(R) (0).The following abbreviations are used Boc
t-butyloxycarbonyl
- Z(OMe)
p-methoxybenzyloxycarbonyl
- MBzl
p-methoxybenzyl
- Acm
acetamidomethyl
- Bzl
benzyl
- Ad
l-adamantyl
- tBu
t-butyl
- TFA
trifluoroacetic acid
- TFMSA
trifluoromethanesulfonic acid
- TMSOTf
trimethylsilyl trifluoromethane sulfonate 相似文献
7.
In vitro and in vivo results are presented demonstrating that superoxide reductase (SOR) from the air-sensitive oral spirochete, Treponema denticola (Td), is a principal enzymatic scavenger of superoxide in this organism. This SOR contains the characteristic non-heme [Fe(His)(4)Cys] active sites. No other metal-binding domain has been annotated for Td SOR. However, we found that Td SOR also accommodates a [Fe(Cys)(4)] site whose spectroscopic and redox properties resemble those in so-called 2Fe-SORs. Spectroscopic comparisons of the wild type and engineered Cys → Ser variants indicate that three of the Cys ligands correspond to those in [Fe(Cys)(4)] sites of "canonical" 2Fe-SORs, whereas the fourth Cys ligand residue has no counterpart in canonical 2Fe-SORs or in any other known [Fe(Cys)(4)] protein. Structural modeling is consistent with iron ligation of the "noncanonical" Cys residue across subunit interfaces of the Td SOR homodimer. The Td SOR was isolated with only a small percentage of [Fe(Cys)(4)] sites. However, quantitative formation of stable [Fe(Cys)(4)] sites was readily achieved by exposing the as-isolated protein to an iron salt, a disulfide reducing agent and air. The disulfide/dithiol status and iron occupancy of the Td SOR [Fe(Cys)(4)] sites could, thus, reflect intracellular redox status, particularly during periods of oxidative stress. 相似文献
8.
Adélia C. O. Cintra Suely V. Sampaio Arni K. Raghuvir José R. Giglio 《Journal of Protein Chemistry》2001,20(5):377-382
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. 相似文献
9.
Human antithrombin III (AT-III) was partially reduced under mild conditions in the absence or presence of low molecular weight heparin. Quantitation of reduced disulfide bonds was facilitated by the application of a water-soluble color reagent, 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid (S-DABIA). The study shows that the three disulfide linkages of AT-III can be sequentially reduced, with Cys8-Cys128 being the most sensitive, followed by Cys21-Cys95, while Cys247-Cys430 is the most resistant to the mild reduction conditions. The rate of reduction of Cys8-Cys128 and Cys21-Cys95 was significantly decreased in the presence of heparin. The reduction of Cys8-Cys128 was also found to correlate quantitatively with the loss of heparin-accelerated antithrombin activity, heparin binding affinity, and heparin-induced fluorescence enhancement. These results suggest that Cys8-Cys128 is required for the integrity of the heparin binding domain of AT-III and support previous findings that lysyl residues surrounding Cys128 (Lys107, Lys114, Lys125, and Lys136) constitute an important part of the heparin binding site in AT-III. 相似文献
10.
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. 相似文献
11.
Gastrin is initially synthesized as a large precursor that requires endoproteolytic cleavage by a prohormone convertase (PC) for bioactivation. Gastric antral G-cells process progastrin at Arg(94)Arg(95) and Lys(74)Lys(75) residues generating gastrin heptadecapeptide (G17-NH(2)). Conversely, duodenal G-cells process progastrin to gastrin tetratriacontapeptide (G34-NH(2)) with little processing at Lys(74)Lys(75). Both tissues express PC1/PC3 and PC2. Previously, we demonstrated that heterologous expression of progastrin in an endocrine cell line that expresses PC1/PC3 and little PC2 (AtT-20) resulted in the formation of G34-NH(2). To confirm that PC1/PC3 was responsible for progastrin processing in AtT-20 cells and capable of processing progastrin in vivo we coexpressed either human wild-type (Lys(74)Lys(75)) or mutant (Arg(74)Arg(75), Lys(74)Arg(75), and Arg(74)Lys(75)) progastrins in AtT-20 cells with two different antisense PC1/PC3 constructs. Coexpression of either antisense construct resulted in a consistent decrease in G34-NH(2) formation. Gastrin mRNA expression and progastrin synthesis were equivalent in each cell line. Although mutation of the Lys(74)Lys(75) site within G34-NH(2) to Lys(74)Arg(75) resulted in the production of primarily G17-NH(2) rather than G34-NH(2), inhibition of PC1/PC3 did not significantly inhibit processing at the Lys(74)Arg(75) site. We conclude that PC1/PC3 is a progastrin processing enzyme, suggesting a role for PC1/PC3 progastrin processing in G-cells. 相似文献
12.
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. 相似文献
13.
Rearrangement of disulfide bonds during the synthesis of alpha-conotoxin GI using PhS(O)Ph/CH(3)SiCl(3) oxidation procedure was observed. We have demonstrated that the protecting scheme (order of acetamidomethyl (Acm) and (t)Bu protecting groups) of the Cys residues as well as the reaction time influenced the ratio of the native and the mispaired compounds, while the temperature of the reaction mixture had no significant effect. However, in all cases the nonnative derivative was produced in high amount. The structure of the isomers was identified by the combination of enzymatic digestion and mass spectrometry measurements. We conclude that the air oxidation followed by the application of Tl(tfa)(3) for the regioselective formation of disulfide bonds leads up to the appropriate compound in the case of the synthesis of alpha-conotoxin GI, while the oxidation procedure using PhS(O)Ph/CH(3)SiCl(3) system resulted in the nonnative disulfide isomer. 相似文献
14.
Sulfhydryl cross-linking poly(ethylene glycol) (PEG)-peptides and glycopeptides were prepared and tested for spontaneous polymerization by disulfide bond formation when bound to plasmid DNA, resulting in stable PEG-peptide and glycopeptide DNA condensates. A 20 amino acid synthetic peptide possessing a single sulfhydryl group on the N-terminal cysteine, with two or five internal acetamidomethyl (Acm)-protected cysteine residues, was reacted with either PEG vinyl sulfone or iodoacetamide tyrosinamide triantennary N-glycan. Following RP-HPLC purification, Acm groups were removed by silver tetrafluoroborate to generate sulfhydryl cross-linking PEG-peptides and glycopeptide that were characterized by either (1)H NMR or LC-MS. Sulfhydryl cross-linking PEG-peptides and glycopeptides were found to bind to plasmid DNA and undergo disulfide cross-linking resulting in stable DNA condensates with potential utility for in vivo gene delivery. 相似文献
15.
16.
Hirudin, the thrombin-specific inhibitor from the leech Hirudo medicinalis, is a single-chain polypeptide (65 amino-acid residues) linked by three disulfide bridges. Localization of the three disulfide bonds could be assigned on the basis of the structures of cystine peptides derived by high performance liquid chromatography separations of thermolysinolytic digest of native hirudin. By characterization of the nine major fragments by amino-acid analysis, N-terminal amino-acid determination and sequence analysis, the following disulfide linkages were identified: Cys6-Cys14, Cys16-Cys28 and Cys22-Cys39. Due to the lack of any closer sequence homology and topological structural homology to other serine proteinase inhibitor proteins, hirudin seems to be unique in its primary structure and hence designates an unknown inhibitor family. 相似文献
17.
Investigation of the structure/activity relationship of human calcitonin gene-related peptide (CGRP) 总被引:6,自引:0,他引:6
J R Tippins V Di Marzo M Panico H R Morris I MacIntyre 《Biochemical and biophysical research communications》1986,134(3):1306-1311
The biological activities of calcitonin gene-related peptide (CGRP) enzymic digest fragments, chemically modified products and beta-CGRP have been compared to that of intact alpha-CGRP on rat isolated paired atria. Tryptic and chymotryptic digests both produced inactive fragments. Acetylation of the N-terminal amino acid (Alanine) or either of Lys 24 or Lys 35, resulted in reduced, but measurable, biological activity. Destruction of the disulphide bridge between Cys 2 and Cys 7 abolished biological activity. Substitution of several amino acids, Asp 3, Val 22 and Asn 25, with Asn, Met and Ser respectively (beta-CGRP), produced a peptide with similar biological activity to alpha-CGRP. 相似文献
18.
A novel receptor for calcitonin gene-related peptide (CGRP) mediates secretion in the rat colon: implications for secretory function in colitis. 总被引:1,自引:0,他引:1
T Esfandyari W K Macnaughton R Quirion S St Pierre J L Junien K A Sharkey 《FASEB journal》2000,14(10):1439-1446
The receptor responsible for CGRP-induced ion transport and permeability was examined in tissues from animals treated 7 days previously with trinitrobenzenesulfonic acid to induce colitis or in controls. CGRP caused a concentration-dependent increase in short circuit current (I(sc), EC(50) 21 nM), which was abolished in chloride-free buffer but was not blocked by CGRP(8-37) or tetrodotoxin (TTX). Amylin and adrenomedullin caused only a modest increase in I(sc). The responses to the linear CGRP(2) receptor agonists [Cys(Et)(2,7)] hCGRPalpha and [Cys(Acm)(2,7)] hCGRPalpha were considerably smaller than the response to CGRP. These responses were abolished in chloride-free buffer and were TTX sensitive. Atropine, doxantrazole, and indomethacin did not block the effects of CGRP or the CGRP(2) agonists. The response to [Cys(Et)(2,7)] hCGRPalpha was not affected by prior desensitization of the CGRP receptor and vice versa. Inflamed rats had a similar secretory response to CGRP (I(sc), EC(50) 15 nM) and [Cys(Et)(2,7)] hCGRPalpha as control tissues, while being hyporesponsive to carbachol. CGRP application increased electrical conductance of inflamed preparations. Taken together, these data suggest that CGRP may play an important role in the maintenance of host defense in colitis through an apparently novel CGRP receptor located on the colonic enterocyte. 相似文献
19.
Synthesis of a membrane protein with two transmembrane regions. 总被引:1,自引:0,他引:1
Takeshi Sato Toru Kawakami Kenichi Akaji Hiroki Konishi Koji Mochizuki Toshimichi Fujiwara Hideo Akutsu Saburo Aimoto 《Journal of peptide science》2002,8(4):172-180
A membrane protein with two transmembrane domains was synthesized by means of the thioester method. The F1F0 ATP synthase subunit c (Sub.c), which consists of 79 amino acid residues (MW 8257), was chosen as a target. For synthetic purposes, two building blocks, Boc-[Lys34(Boc)]-Sub.c(1-38)-SCH2CH2CO-Ala and Sub.c(39-79), were synthesized via solid-phase methods using Boc chemistry. RP-HPLC purification conditions for the transmembrane peptide were examined. As a result, a combination of a mixture of formic acid, 1-propanol and water with a phenyl column was found to be useful for separating the transmembrane peptide. The purified building blocks were condensed in DMSO in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), N,N-diisopropylethylamine to give the product, Sub.c, after removal of Boc groups (yield 16%). The yield of the condensation reaction could be improved to 23% by raising the reaction temperature to 50 degrees C, and to 26% when a mixture of chloroform and methanol was used as a solvent. 相似文献
20.
Hong SY Sohn YD Chung KH Kim DS 《Biochemical and biophysical research communications》2002,293(1):530-536
Saxatilin is a 7.7 kDa disintegrin that belongs to a family of homologous protein found in several snake venoms. Six disulfide bond locations of the disintegrin were determined by enzymatic cleavage and matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF). Functional implications of the disulfide bonds related to the biological activity of saxatilin were investigated with recombinant protein species produced by site-directed mutagenesis of saxatilin. Several lines of experimental evidence indicated that three disulfide bonds, Cys21-Cys35, Cys29-Cys59, and Cys47-Cys67, of the disintegrin are closely associated with its biological function such as its ability to block the binding of integrin GPIIb-IIIa and alpha(v)beta(3) with fibrinogen and extracellular matrix. Those disulfide linkages were also revealed to be important for maintaining the functional structure of the protein molecule. On the other hand, the disulfide bridges of Cys6-Cys15 and Cys8-Cys16 do not appear to be critical for the molecular structure and function of saxatilin. 相似文献