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1.
VP1 is the major coat protein of murine polyomavirus and forms virus-like particles (VLPs) in vitro. VLPs consist of 72 pentameric VP1 subunits held together by a terminal clamp structure that is further stabilized by disulfide bonds and chelation of calcium ions. Yeast-derived VLPs (yVLPs) assemble intracellularly in vivo during recombinant protein production. These in vivo assembled yVLPs differ in several properties from VLPs assembled in vitro from bacterially produced pentamers. We found several intermolecular disulfide linkages in yVLPs involving 5 of the 6 cysteines of VP1 (Cys115–Cys20, Cys12–Cys20, Cys16–Cys16, Cys12/ Cys16–Cys115, and Cys274–Cys274), indicating a highly coordinated disulfide network within the in vivo assembled particles involving the N-terminal region of VP1. Cryoelectron microscopy revealed structured termini not resolved in the published crystal structure of the bacterially expressed VLP that appear to clamp the pentameric subunits together. These structural features are probably the reason for the observed higher stability of in vivo assembled yVLPs compared with in vitro assembled bacterially expressed VLPs as monitored by increased thermal stability, higher resistance to trypsin cleavage, and a higher activation enthalpy of the disassembly reaction. This high stability is decreased following disassembly of yVLPs and subsequent in vitro reassembly, suggesting a role for cellular components in optimal assembly.  相似文献   

2.
The crucial step of folding of recombinant proteins presents serious challenges to obtaining the native structure. This problem is exemplified by insulin‐like growth factor (IGF)‐I which when refolded in vitro produces the native three‐disulfide structure, an alternative structure with mispaired disulfide bonds and other isomeric forms. To investigate this phenomenon we have examined the refolding properties of an analog of IGF‐I which contains a 13‐amino acid N‐terminal extension and a charge mutation at position 3 (Long‐ [Arg3]IGF‐I). Unlike IGF‐I, which yields 45% of the native structure and 24% of the alternative structure when refolded in vitro, Long‐[Arg3]GF‐I yields 85% and 10% of these respective forms. To investigate the interactions that affect the refolding of Long‐[Arg3]IGF‐I and IGF‐I, we acid‐trapped folding intermediates and products for inclusion in a kinetic analysis of refolding. In addition to non‐native intermediates, three native‐like intermediates were identified, that appear to have a major role in the in vitro refolding pathway of Long‐[Arg3]IGF‐I; a single‐disulfide Cys18–Cys61 intermediate, an intermediate with Cys18–Cys61 and Cys6–Cys48 disulfide bonds and another with Cys18–Cys61 and Cys47–Cys52 disulfide bonds. Furthermore, from our kinetic analysis we propose that the Cys18‐Cys61, Cys6‐Cys48 intermediate forms the native structure, not by the direct formation of the last (Cys47‐Cys52) disulfide bond, but by rearrangement via the Cys18–Cys61 intermediate and a productive Cys18–Cys61, Cys47–Cys52 intermediate. In this pathway, the last disulfide bond to form involves Cys6 and Cys48. Finally, we apply this pathway to IGF‐I and conclude that the divergence in the in vitro folding pathway of IGF‐I is caused by non‐native interactions involving Glu3 that stabilize the alternative structure. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 693–703, 1999.  相似文献   

3.
Eyestalks of the palinuran species Jasus lalandii and Panulirus homarus, and the brachyuran species Carcinus maenas, were examined with antisera raised against purified crustacean hyperglycaemic hormone (cHH) of the astacidean species Homarus americanus and Procambarus bouvieri, as well as the brachyuran species Cancer pagurus. Other antisera used in this investigation were raised against purified moult-inhibiting hormone (MIH) of C. pagurus and vitellogenesis-inhibiting hormone (VIH) of H. americanus. Positive immunoreactions to all the antisera were localised in perikarya of the X-organ and the axon terminals in the sinus gland of all the crustaceans investigated. These results illustrate the existence of an immunological similarity, detectable at the immunocytochemical level, between the cHH/MIH/VIH neurohormones of the Astacidae, Palinura and Brachyura infraorders. Furthermore, results from consecutive tissue sections indicate that cHH, MIH and VIH are co-localised in a subpopulation of X-organ neurons.  相似文献   

4.
Human DNA polymerases mu (Polµ) and lambda (Polλ) are X family members involved in the repair of double-strand breaks in DNA during non-homologous end joining. Crucial abilities of these enzymes include bridging of the two 3′ single-stranded overhangs and trans-polymerization using one 3′ end as primer and the other as template, to minimize sequence loss. In this context, we have studied the importance of a previously uncharacterised sequence (‘brooch’), located at the N-terminal boundary of the Polß-like polymerase core, and formed by Tyr141, Ala142, Cys143, Gln144 and Arg145 in Polµ, and by Trp239, Val240, Cys241, Ala242 and Gln243 in Polλ. The brooch is potentially implicated in the maintenance of a closed conformation throughout the catalytic cycle, and our studies indicate that it could be a target of Cdk phosphorylation in Polµ. The brooch is irrelevant for 1 nt gap filling, but of specific importance during end joining: single mutations in the conserved residues reduced the formation of two ended synapses and strongly diminished the ability of Polµ and polymerase lambda to perform non-homologous end joining reactions in vitro.  相似文献   

5.
Hen ovalbumin contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryl groups (Cys11,Cys30,Cys367, and Cys382) in a single polypeptide chain of 385 amino acid residues. To investigate whether or not such a structure is shared by related avian species, the contents of disulfide-involved half-cystine residues and their positions in the primary structure of ovalbumins from five species were compared with those of hen ovalbumin. Ovalbumins were alkylated with a fluorescent dye, IAEDANS, under disulfide-reduced and disulfide-intact conditions and digested with a number of proteolytic enzymes. The sequences were deduced from peptides containing half-cystine residues labeled with the fluorescent dye. The results showed that the number of free cysteine sulfhydryl groups of ovalbumins was different among the species, three for guinea fowl and turkey (Cys11, Cys367, and Cys382); and two for Pekin duck, mallard duck, and Emden goose (Cys11 and Cys331). On the other hand, a single intrachain disulfide bond could be identified from ovalbumins of five species using a combination of peptide mapping and N-terminal amino acid sequencing analysis under reduced and non-reduced conditions, in which the intrachain disulfide bond was like that of hen ovalbumin (Cys73-Cys120). The results also indicated that the variations in amino acid sequences on these peptides containing half-cystine residues bear a close relationship with the phylogeny of the six species.  相似文献   

6.
Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type.  相似文献   

7.
A full-length cDNA coding lipoprotein lipase (LPL) was cloned from liver of adult common carp (Cyprinus carpio Var. Jian) by RT-PCR and rapid amplification of cDNA ends (RACE) approaches. The cDNA obtained was 2,411 bp long with a 1,524 bp open reading frame (ORF) encoding 507 amino acids. This amino acid sequence contains two structural regions: N-terminus (24–354 residues) and C-terminus (355–507 residues). Before N-terminus, 1–23 residues is signal peptide, 6–23 residues is transmembrance helix. At N-terminus, some conversed functional sites were found, including two N-linked glycosylation sites Asn41 and Asn88; one catalytic triad Ser174, Asp198 and His283; one conserved heparin-binding site Arg321 to Arg324 (RKNR); eight cysteines residues Cys69 and Cys82, Cys258 and Cys281, Cys306 and Cys325, Cys317 and Cys320 which are involved in four disulfide bridges; one polypeptide “lid” that participates in substrate specificity. At C-terminus, Asn401 is another N-linked glycosylation site, and Trp434 and Trp435 (WW) is lipid-binding site. The amino acid sequence has a high similarity, and shows similar structural features to LPL of other species. Tissue distribution of LPL mRNA in liver, head kidney, mesenteric adipose tissue, heart and white muscle of common carp was analyzed by semi-quantitative RT-PCR method using β-actin gene as internal control. The result showed that the expressions of LPL mRNA were detected in all examined tissues of common carp. The expression levels of LPL in the mesenteric adipose tissue was highest among these tissues, following in liver and head kidney, and the lowest expression was found in heart and white muscle.  相似文献   

8.
Peroxiredoxins (Prxs) are thiol peroxidases that scavenge various peroxide substrates such as hydrogen peroxide (H2O2), alkyl hydroperoxides and peroxinitrite. They also function as chaperones and are involved in signal transduction by H2O2 in eukaryotic cells. The genome of Aquifex aeolicus, a microaerophilic, hyperthermophilic eubacterium, encodes four Prxs, among them an alkyl hydroperoxide reductase AhpC2 which was found to be closely related to archaeal 1-Cys peroxiredoxins. We determined the crystal structure of AhpC2 at 1.8?Å resolution and investigated its oligomeric state in solution by electron microscopy. AhpC2 is arranged as a toroid-shaped dodecamer instead of the typically observed decamer. The basic folding topology and the active site structure are conserved and possess a high structural similarity to other 1-Cys Prxs. However, the C-terminal region adopts an opposite orientation. AhpC2 contains three cysteines, Cys49, Cys212, and Cys218. The peroxidatic cysteine CP49 was found to be hyperoxidized to the sulfonic acid (SO3H) form, while Cys212 forms an intra-monomer disulfide bond with Cys218. Mutagenesis experiments indicate that Cys212 and Cys218 play important roles in the oligomerization of AhpC2. Based on these structural characteristics, we proposed the catalytic mechanism of AhpC2. This study provides novel insights into the structure and reaction mechanism of 1-Cys peroxiredoxins.  相似文献   

9.
Among the main classes of cysteine-stabilized antimicrobial peptides, the snakin/GASA family has not yet had any structural characterization. Through the combination of ab initio and comparative modeling with a disulfide bond predictor, the three-dimensional structure prediction of snakin-1 is reported here. The structure was composed of two long α-helices with a disulfide pattern of CysI-CysIX, CysII-CysVII, CysIII-CysIV, CysV-CysXI, CysVI-CysXII and CysVIII-CysX. The overall structure was maintained throughout molecular dynamics simulation. Snakin-1 showed a small degree of structural similarity with thionins and α-helical hairpins. This is the first report of snakin-1 structural characterization, shedding some light on the snakin/GASA family.  相似文献   

10.
The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C-terminally truncated GLP-1R. Single cysteines were initially substituted with alanine, and functionally redundant cysteines were subsequently changed simultaneously. Our results indicate that Cys174, Cys226, Cys296 and Cys403 are important for the GLP-1-mediated response, whereas Cys236, Cys329, Cys341, Cys347, Cys438, Cys458 and Cys462 are not. Extensive deletions were made in the C-terminal tail of GLP-1R in order to determine the limit for truncation. As for other family B GPCRs, we observed a direct correlation between the length of the C-terminal tail and specific binding of 125I-GLP-1, indicating that the membrane proximal part of the C-terminal is involved in receptor expression at the cell surface. The results show that seven cysteines and more than half of the C-terminal tail can be removed from GLP-1R without compromising GLP-1 binding or function.  相似文献   

11.
Chen X  Wang L  Wang H  Chen H  Zhou M  Chen T  Shaw C 《Peptides》2011,32(1):26-30
Extensive studies on bradykinin-related peptides (BRPs) generated from plasma kininogens in representative species of various vertebrate taxa, have confirmed that many amphibian skin BRPs reflect those present in putative vertebrate predators. For example, the (Val1, Thr6)-bradykinin, present in the defensive skin secretions of many ranids and phyllomedusines, can be generated from plasma kininogens in colubrid snakes—common predators of these frogs. Here, we report the presence of (Arg0, Trp5, Leu8)-bradykinin in the skin secretion of the European edible frog, Pelophylax kl. esculentus, and have found it to be encoded in single copy by a kininogen with an open-reading frame of 68 amino acid residues. This peptide is the archetypal bony fish bradykinin that has been generated from plasma kininogens of the bowfin (Amia calva), the long-nosed gar (Lepisosteus oseus) and the rainbow trout (Onchorhynchus mykiss). More recently, this peptide has been shown to be encoded within cloned kininogens of the Atlantic cod (Gadus morhua) spotted wolf-fish (Anarichas minor), zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and Northern pike (Esox lucius). The latter species is regarded as a major predator of P. kl. esculentus. Synthetic (Arg0, Trp5, Leu8)-bradykinin was previously reported as having multiphasic effects on arterial blood pressure in conscious trout and here we have demonstrated that it can antagonize the relaxation in rat arterial smooth muscle induced by canonical mammalian bradykinin. The discovery of (Arg0, Trp5, Leu8)-bradykinin in the defensive skin secretion of this amphibian completes the spectrum of vertebrate taxon-specific BRPs identified from this source.  相似文献   

12.
Our previous study revealed a mono-ADP-ribosyltransferase mediated in vitro mono-ADP-ribosylation of IC3 peptide, a peptide with sequence corresponded to third intracellular loop of glucagon like-peptide-1 (GLP-1) receptor. Furthermore, Arg348 was shown to be modified amino acid residue although its mutation did not eliminate mono-ADP-ribosylation completely. In order to further study the signaling mechanisms of GLP-1 receptor, we took on lease a possibility that an alternative site of enzymatic modification exist so mono-ADP-ribosylation of Cys341 was hypothesized. The results confirmed both Arg348 and Cys341 as a site of mono-ADP-ribosylation where Arg348 is modified predominantly. Sum of mono-ADP-ribosylation rate of both single IC3 mutants coincided with IC3 rate. What is in vivo role of Cys341 mono-ADP-ribosylation is entirely speculative but our study represents an important step toward a complete understanding of signaling via GLP-1 receptor.  相似文献   

13.
14.
We examined the taxonomic position of Leptosphaerulina japonica, a parasitic fungus on male strobili of Japanese cedar (Cryptomeria japonica) using morphology and molecular phylogeny. Because neither specimens nor illustrations were designated in the original paper, this name is neotypified with a fresh specimen collected from the type locality, Yamagata, Japan. Leptosphaerulina japonica has asci produced in a broad layer arising from the entire base of the locule, stipitate acervuli, and a hormonema-like synanamorph. These characteristics are not typical of the genus Leptosphaerulina (Didymellaceae, Pleosporales). In a molecular phylogenetic analysis of ITS sequences, L. japonica was placed into the Dothideaceae/Dothioraceae, Dothideales. Although muriform ascospores and an anamorph with long stipitate acervuli have not been reported for species in Sydowia, the other morphology and phylogeny support placement of L. japonica in that genus. Thus, a new combination, Sydowia japonica, is proposed.  相似文献   

15.
Neurotensin (NT) analogs, modified at Arg8 and Arg9, were used to assess the role of Arg in NT binding to mast cells. [D-Arg8]- and [D-Arg9]-NT bound 4–5 times better than NT, whereas [D-Arg8,9]-NT had the same binding affinity as NT. Binding of [Ala8]-NT was not parallel to NT and exhibited a dissociation constant 38-fold lower than NT while [Ala9]-NT had 32% binding. C-terminal peptides, NT8–13 and NT9–13, had about 65% binding. These data suggest that Arg8 plays a greater role than Arg9 in the binding to mast cell NT receptors. Reduction of the disulfide bond in [Cys2,13]-NT produced an analog 4-times more potent than NT, while the cyclized form had only 3% binding. Thus, a linear peptide with a free C-terminus appears to be required for binding.  相似文献   

16.
17.
Transfer RNAs (tRNA) are important molecules that involved in protein translation machinery and acts as a bridge between the ribosome and codon of the mRNA. The study of tRNA is evolving considerably in the fields of bacteria, plants, and animals. However, detailed genomic study of the cyanobacterial tRNA is lacking. Therefore, we conducted a study of cyanobacterial tRNA from 61 species. Analysis revealed that; cyanobacteria contain thirty-six to seventy-eight tRNA gens per genome that encodes for 20 tRNA isotypes. The number of iso-acceptors (anti-codons) ranged from thirty-two to forty-three per genome. tRNAIle with anti-codon AAU, GAU, and UAU was reported to be absent from the genome of Gleocapsa PCC 73,106 and Xenococcus sp. PCC 7305. Instead, they were contained anti-codon CAU that is common to tRNAMet and tRNAIle as well. The iso-acceptors ACA (tRNACys), ACC (tRNAGly), AGA, ACU (tRNASer), AAA (tRNAPhe), AGG (tRNAPro), AAC (tRNAVal), GCG (tRNAArg), AUG (tRNAHis), and AUC (tRNAAsp) were absent from the genome of cyanobacterial lineages studied so far. A few of the cyanobacterial species encode suppressor tRNAs, whereas none of the species were found to encode a selenocysteine iso-acceptor. Cyanobacterial species encode a few putative novel tRNAs whose functions are yet to be elucidated.  相似文献   

18.
The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg334, Arg396 and Arg638 were directly assigned to the ETF and ii) Arg198 was assigned as the only tryptic site to the LTF. Arg671, Lys712/Lys713 and Lys728 were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl2, EGTA or AlF4 strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg1002 as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.  相似文献   

19.
珠江口附近海区甲壳类动物的区系特征及其分布状况   总被引:4,自引:0,他引:4  
根据2006年8月(夏季)、2006年10月(秋季)、2006年12月(冬季)和2007年4月(春季)珠江口附近海区的底拖网调查资料,分析了该海域甲壳类动物的区系特征和分布状况.结果表明:本次调查共采获甲壳类动物54种,分隶于2目17科25属,其中,虾类22种、蟹类22种、虾蛄类10种;该海域甲壳类动物多数为热带 亚热带的暖水性种类,少数为广温性种类,未出现暖温性和冷温性种类;广盐性的种类最多,其次为高盐性种类,低盐性种类最少;大多数种类属于印度洋 西太平洋区系;调查海域的甲壳类动物种类与东海、菲律宾海、印度尼西亚海、日本海关系较密切,与黄海、渤海、朝鲜海关系较疏远.调查海域甲壳类动物的优势种为周氏新对虾、口虾蛄、武士蟳、红星梭子蟹、猛虾蛄、锈斑蟳、日本蟳、长叉口虾蛄、中华管鞭虾、三疣梭子蟹和逍遥馒头蟹.出现的种类数以秋季最多(33种),春季最少(26种).甲壳类资源密度在水深0~40 m水域较高,在水深10~20 m最密集.调查海域甲壳类动物的平均资源密度为99.60 kg·km-2,以夏季最高(198.93 kg·km-2)、春季最低(42.35 kg·km-2);组成甲壳类动物的3个类群中,蟹类的资源密度最高(41.81 kg·km-2),其次是虾类(38.91 kg·km-2),虾蛄类最低(18.88 kg·km-2);各类群资源密度存在明显的季节变化,虾类在夏季最高(120.32 kg·km-2)、春季最低(0.67 kg·km-2),蟹类和虾蛄类则均在冬季最高(62.01和29.49 kg·km-2)、秋季最低(24.64和6.30 kg·km-2).  相似文献   

20.
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