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PubMed1997

Full-length and N-terminally truncated chicken intestinal diazepam-binding inhibitor. Purification, structural characterization and influence on insulin release.

Two forms of diazepam-binding inhibitor (DBI) have been purified from chicken intestine and identified as the intact avian polypeptide (residues 1-86) and a truncated variant (residues 35-86). At 10 nM concentration, both the intact and the truncated peptide suppress in vitro-monitored glucose-induced insulin release by 50 (p < 0.02) and 64% (p < 0.01) respectively. The truncation starts at a segment. -Thr-Val-Gly-Asp-, that is strictly conserved between characterized DBI species, indicating special restrictions on the structure. However, overall DBI conservation appears to be complex. A number of differently bioactive fragments with separate processings and tissue distributions have been observed, suggesting multiple functions of DBI and its sub-segments.

Amino Acid Sequence↗
PubMed2000

Structure at 1.44 A resolution of an N-terminally truncated form of the rat serum complement C3d fragment.

Complement component C3 plays a key role in the complement-mediated immune defence, and occupies a central position within the complement cascade system. One of its degradation products, C3dg, was purified from rat serum and crystallised in two different crystal forms as N-terminally truncated fragment. Despite the truncation and the lack of a significant portion of the N-terminus as compared to C3d, the structure of the fragment is highly similar to that of recombinant human C3d (Nagar et al., Science 280 (1998) 1277-1281). Structural details of the reactive site have been obtained, suggesting a possible mode of thioester bond formation between Cys-1010 and Gln-1013 and thioester bond cleavage in the transacylation reaction involving His-1126. The truncation at the N-terminus of C3d leads to the exposure of a surface of the molecule that favours dimerisation, so that in both crystal forms, the fragment is present as a dimer, with monomers related by a two-fold axis.

Animals↗
PubMed1998

1H and 13C NMR studies of a truncated heme domain from Chlorella vulgaris nitrate reductase: signal assignment of the heme moiety.

A water soluble truncated heme domain (a tetramer of MW = 45 kDa) of the tetrameric nitrate reductase complex from the green alga Chlorella vulgaris has been overexpressed and purified. This truncated heme domain with four identical subunits has a high redox potential (midpoint potential E1/2 = +16 mV) as compared with other heme-containing flavoproteins. We have undertaken a determination of the detailed configuration of the heme moiety in order to understand the unique electrochemical property of the heme moiety of this enzyme. We report here the study of the heme prosthetic group of the truncated heme domain by the use of 2D 1H and 13C NMR techniques. A complete signal assignment of the heme has been achieved. Our observations suggest that the heme configuration is similar to that of the crystal structure of the membrane-bound bovine liver cytochrome b5.

Animals↗
PubMed2002

Immunization with a DNA vaccine expressing a truncated form of varicella zoster virus glycoprotein E.

The gE glycoprotein of varicella zoster virus (VZV) is involved with cell entry and it is the most abundant glycoprotein produced in VZV-infected cells. It is also the first glycoprotein to be recognized by the immune system and induces neutralizing antibodies and cellular immunity. We have shown previously that immunization with a DNA vaccine encoding full length gE induces high antibody titres in BALB/c mice. In this study, we engineered a truncated form of gE to facilitate secretion of the glycoprotein, which is thought to increase the quantity of antigen available for B cells to mount an immune response. This hypothesis was tested by using inverse PCR mutagenesis (IPCRM) to engineer a mutated form of gE that was secreted from the cell. This construct was then evaluated as a potential DNA vaccine. Following immunization studies, the magnitude of the immune response induced with the mutant form of gE was found to be similar to that induced by membrane bound protein. This finding suggests that, in the case of VZV, a DNA vaccine expressing a secreted protein has no advantage over one expressing a membrane bound protein. However, mice immunized with the truncated form of gE (gED) displayed responses favouring IgG1 (Th2) in comparison with mice immunized with the full length gE construct, which generated an IgG2a (Th1) response. This observation indicates that immunization with a truncated form of a gene may induce immune modulation, a phenomenon that should be taken into account for the design of vaccines.

Animals↗
PubMed1998

Difference in dominant negative activities between mutant thyroid hormone receptors alpha1 and beta1 with an identical truncation in the extreme carboxyl-terminal tau4 domain.

Although different expression patterns of thyroid hormone receptor (TR) alpha1 and beta1 have been reported, no essential distinction has been established in their functions. Unlike the TR beta gene, a mutation in the TR alpha1 gene has never been found in patients with resistance to thyroid hormone (RTH). Previously we found a mutant TR beta with an 11-carboxyl (C)-terminal amino acid truncation (betaF451X) in a girl with severe RTH. BetaF451X is a natural mutant with disruption of the transactivation domain, tau4, and it had very strong dominant negative activities. Based on the fact that the 46 amino acid sequence in the extreme C-terminal region is identical in TR alpha1 and TR beta, except for a C-terminal three amino acid extension of TR alpha1, we constructed a mutant TR alpha1 (alphaF397X) with the identical C-terminal truncation to betaF451X, to study functional differences between TR alpha1 and beta1. Both betaF451X and alphaF397X had negligible T3 binding and transcriptional activities even with 1 microM T3. The dominant negative activities of the mutant TRs were remarkable and T3 response element (TRE)-dependent. Co-expression of betaF451X decreased the CAT activity of either wild-type TR alpha1 or beta1 at 100 nM T3 by approximately 90% on the TRE-pal2 and 70% on DR4. AlphaF397X inhibited the transcriptional activities of both wild-type TR alpha1 and beta1 by approximately 50% on TRE-pal2 and by 60% on DR4. The dominant negative potency of betaF451X was significantly stronger than that of alphaF397X on the TRE-pal2, -DR4 and chicken lysozyme silencer F2, but similar on TRE-myosin heavy chain alpha and malic enzyme. No partiality for the TR subtypes was found in the dominant negative effects of betaF451X and alphaF397X. Co-expression with RXR enhanced the dominant negative effects of alphaF397X, but not of betaF451X. The results indicate that there are different dominant negative properties between alphaF397X and betaF451X, which are TRE-dependent, despite their identical C-terminal truncation. Deletion in the tau4 domain might affect the receptor structures of TR alpha1 and beta1 differently.

Animals↗
PubMed1996

The effect of truncated infections with Ostertagia ostertagi on the development of acquired resistance in calves.

The relative contribution of the third (L3), fourth (L4) and adult stages of Ostertagia ostertagi to the development of immunity was assessed in calves which were either continuously infected during 21 weeks or subjected to infections truncated by anthelmintic treatment at the L3 or L4 stage. A fourth group remained uninfected (control group). Faecal samples and blood samples were collected weekly for faecal egg counts and determination of pepsinogen and antibody levels. Only the continuously infected animals showed positive egg counts, which fell towards the end of the primary infection period. Pepsinogen and antibody levels remained high in the continuously infected group until the end of the primary infection period. At that time, they were significantly higher compared to the control calves, with intermediate values in the truncated infection groups. After the 21 weeks primary infection period all animals were dewormed. To evaluate the protection provided by the different immunisation protocols, all animals were challenged 1 week later with 156000 Ostertagia L3, spread over 12 consecutive days. The marked reduction in egg counts following challenge infection indicated a certain degree of immunity in the continuously infected calves, which was confirmed at necropsy by the reduced worm burdens, the high percentage of inhibited early L4 larvae, the reduced size of the adult worms and the higher numbers of mucosal mast cells in this group. Numbers of globule leucocytes and eosinophils were not significantly different from the control group. Infections truncated by anthelmintic treatment elicited poor development of immunity as shown by the egg output after the challenge infection and the percentages of arrested larvae and the lengths of adult worms which were intermediate to those of the continuously infected calves and control animals.

Animals↗
PubMed2002

C-terminal truncation of alpha 1,6-fucosyltransferase from Rhizobium sp. does not annul the transferase activity of the enzyme.

Recently we have over-expressed the enzyme alpha 1,6-fucosyltransferase from Rhizobium sp. in Escherichia coli. In this heterologous system the enzyme was mainly expressed as inclusion bodies and the one that was expressed soluble showed a short-lasting activity in solution due to precipitation of the protein. A structural analysis of the sequence using the TMpred program predicted a highly hydrophobic region of 19 aa close to the C-terminal of the protein. In order to investigate the influence of this region on the formation of inclusion bodies and the precipitation from solution, we cloned a truncated version of the protein where a C-terminal fragment of 65 aa, including the predicted transmembrane-like region, was removed. The resulting protein was expressed in a soluble form without formation of inclusion bodies. The truncated protein catalyzed the transfer of a fucopyranosyl moiety from GDP-beta-L-Fucose to chitobiose. Comparison of the acceptor specificity between the truncated alpha 1,6-fucosyltransferase and the wild-type enzyme, showed a similar behavior for both enzymes. Our results indicate that the active center is not located in the C-terminal extreme of the protein in contrast to the case of the mammalian glycosyltransferases. Also, these results indicate that the alpha-6-motif III is not directly involved in the catalytic activity of the enzyme.

Amino Acid Sequence↗
PubMed1999

Incremental truncation as a strategy in the engineering of novel biocatalysts.

The application and success of combinatorial approaches to protein engineering problems have increased dramatically. However, current directed evolution strategies lack a combinatorial methodology for creating libraries of hybrid enzymes which lack high homology or for creating libraries of highly homologous genes with fusions at regions of non-identity. To create such hybrid enzyme libraries, we have developed a series of combinatorial approaches that utilize the incremental truncation of genes, gene fragments or gene libraries. For incremental truncation, Exonuclease III is used to create a library of all possible single base-pair deletions of a given piece of DNA. Incremental truncation libraries (ITLs) have applications in protein engineering as well as protein folding, enzyme evolution, and the chemical synthesis of proteins. In addition, we are developing a methodology of DNA shuffling which is independent of DNA sequence homology.

Amino Acid Motifs↗
PubMed2001

Efficient generation of the cartesian coordinates of truncated icosahedron and related polyhedra.

Efficient algorithms for deriving the analytical expressions of the rectangular coordinates of the vertices of regular polyhedra and truncated icosahedron inscribed in a cube is described and the results are exposed. Various characteristic quantities of the geometrical structure of truncated icosahedron are obtained. Kaleidoscopes for projecting the truncated icosahedron are discussed.

Carbon↗
PubMed2000

Evidence for a recent mutation giving rise to a truncated copy of a cysteine proteinase gene in Leishmania pifanoi.

We have previously identified and characterized two amastigote-specific cysteine proteinases of Leishmania pifanoi. The slightly different isoforms of the more abundant proteinase are coded by a gene family of approximately 20 gene copies, that contain a C-terminal extension characteristic of cysteine proteinases of trypanosomatids. In this gene family, we have detected a copy that codes for a truncated form of this proteinase, lacking the C-terminal extension. Interestingly, when the deletion of a nucleotide that creates a stop codon causing this truncation is disregarded, the translated sequence gives rise to a divergent C-terminal extension that has many conserved amino acids when compared to Leishmania and Trypanosome, suggesting that a recent mutation led to the truncation.

Amino Acid Sequence↗
PubMed2002

Effect of oligonucleotide truncation on single-nucleotide distinction by solid-phase hybridization.

Oligonucleotide microarrays are used to analyze target sequences on the basis of differences in hybridization stability between matched and mismatched probe-target duplexes. DNA microarray manufacture via photolithographic synthesis generates a minority of full-length oligonucleotide probes along with a series of 5'-truncated contaminants. In a model experiment, we now investigate the effect of truncated oligonucleotides on the ability to distinguish target sequence variants that differ in a single nucleotide position. A series of oligonucleotides, mixed in proportions simulating stepwise synthetic yields of between 82 and 100%, were bound to a solid support and allowed to hybridize to a target molecule. The extent of hybridization was monitored over a range of temperatures via the fluorescence of a double-strand-specific dye. The discriminatory power of pure oligonucleotide probes was found to be significantly greater than that of a population of truncated probes, but only over a limited temperature interval. We conclude that at optimal temperatures greater oligonucleotide quality can improve the performance of oligonucleotide hybridization microarrays.

Animals↗
PubMed1995

Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

Base Sequence↗
PubMed1993

Truncation of the D2 protein in Synechocystis sp. PCC 6803: a role of the C-terminal domain of D2 in photosystem II function and stability.

Termination and deletion mutations were introduced near the C-terminal end of the D2 protein in the cyanobacterium Synechocystis sp. PCC 6803 in order to determine the role of the large hydrophilic C-terminal domain of D2 in the function and stability of photosystem II (PS II). The loss of 57 residues from the C-terminal end of D2 (most of the hydrophilic tail) resulted in the loss of D2 and PS II reaction centers from thylakoids. Truncation of 16, 15, 14, or 13 amino acid residues from the C-terminus of D2 resulted in a virtual disappearance of oxygen evolution, a loss of photoautotrophic growth, and a decrease in the number of PS II centers in thylakoids. The loss of 11 C-terminal amino acid residues led to a photoautotrophic mutant that grew at one-half the rate of the wild type under photoautotrophic conditions and that showed a progressive loss of oxygen evolution at high light intensity. Truncation of 9 residues from D2 led to a virtual loss of CP43, presumably because of interference of the mutation with the overlapping ribosome-binding site for psbC translation. To delete smaller portions of D2 and yet not interfere with psbC expression, various deletions were made between the tenth and twentieth amino acid residues from the C-terminal end of D2, resulting in the loss of 8, 7, 4, 3, and 2 residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Role of the amino-terminal domain in regulating interactions of annexin I with membranes: effects of amino-terminal truncation and mutagenesis of the phosphorylation sites.

Phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of bovine or human annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold (Wang, W., & Creutz, C. E. (1992) Biochemistry 31, 9934-9936). In the present study three forms of human annexin I truncated in the amino terminus at residue Trp-12, Lys-26, or Lys-29 exhibit dramatic differences in their sensitivities to calcium in a chromaffin granule aggregation assay, while the [Ca2+](1/2)max values for binding of the truncated proteins to granule membranes are similar. Cleavage at Trp-12 causes a 3-fold decrease in calcium sensitivity in the membrane aggregation assay, while cleavage at Lys-26 causes a 4-fold enhancement of calcium sensitivity. In contrast, cleavage at Lys-29 results in virtually no change in calcium sensitivity. Mutagenic substitution with negatively charged amino acids of Ser-27, a site for phosphorylation by protein kinase C, or Tyr-21, a site for phosphorylation by the epidermal growth factor receptor kinase, mimics the inhibition of granule-aggregating activity seen with phosphorylation by protein kinase C. When bovine chromaffin cells are stimulated to secrete by nicotine, annexin I is phosphorylated in the amino terminus. Thr-24 and Ser-28, which are sites for phosphorylation by protein kinase C in vitro, are two of the sites phosphorylated in vivo in stimulated chromaffin cells. These data demonstrate that the ability of annexin I to promote membrane aggregation is highly sensitive to changes in the structure of the N-terminal domain of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Effects of carboxyl-terminal truncation on the stability and G protein-coupling activity of bovine rhodopsin.

A number of studies have suggested that G protein-coupled receptors possess domains within the carboxyl terminus that are important for the catalytic activation of G proteins. To define these regions, truncation mutants were generated in the cDNA of bovine rhodopsin, the receptor responsible for visual signal transduction in the retinal rod cell. The mutants were expressed in HEK-293 cells and analyzed for their ability to bind the chromophore, 11-cis-retinal, and for activating Gt, the G protein of the rod cell regulated by rhodopsin. Removal of 38 carboxyl-terminal amino acids resulted in the production of a mutant (K311 stop) that does not bind 11-cis-retinal, has an abnormal pattern of glycosylation, and does not catalyze light-dependent binding of GTP gamma S to Gt, suggesting that it is unable to fold properly during biogenesis. However, a truncation mutant with only five additional amino acids (C316stop) coupled normally to Gt, using membranes from transfected cells, despite the fact that it lacked the "fourth cytoplasmic loop" formed by palmitoylation of cysteines-322 and -323. When C316stop is extracted from the membrane with detergent, only a fraction is able to bind 11-cis-retinal, but the fraction that binds retinal activates Gt normally. In contrast, detergent-solubilized wild-type rhodopsin and K325stop (a truncation mutant with the longest carboxyl terminus) both bind retinal and activate Gt normally. These data suggest that the proximal region of the carboxyl terminus is critical for the proper folding and stability of the rhodopsin molecule and that amino acids Cys316 to Ala348 are not necessary for the activation of Gt.

Amino Acid Sequence↗
PubMed1990

Characterization of ATP and calmodulin-binding properties of a truncated form of Bacillus anthracis adenylate cyclase.

The Bacillus anthracis cya gene encodes a calmodulin-dependent adenylate cyclase. A deletion cya gene product obtained by removing 261 codons at the 5' end was expressed in a protease-deficient lon- E. coli strain and purified to homogeneity. This truncated enzyme (CYA 62) exhibits catalytic and calmodulin-binding properties similar to the properties of wild-type adenylate cyclase from B. anthracis culture supernatants, i.e., a kcat of 1100 s-1 at 30 degrees C and pH 8, an apparent Km for ATP of 0.25 mM, and a Kd for bovine brain calmodulin of 23 nM. The calmodulin-binding domain of the CYA 62 truncated enzyme was labeled with a cleavable radioactive photoaffinity cross-linker coupled to calmodulin. The labeled CYA 62 protein was then cleaved with cyanogen bromide and N-chlorosuccinimide. We show that the calmodulin-binding domain of B. anthracis adenylate cyclase is located within the last 150 amino acid residues of the protein. A further deletion at the 3' end of the CYA 62 coding sequence yielded an adenylate cyclase species (CYA 57) lacking 127 C-terminal amino residues. CYA 57, still sensitive to activation by high concentrations of calmodulin, exhibits less than 0.1% of the specific activity of CYA 62. Binding of 3'dATP (a competitive inhibitor) to CYA 62 was determined by equilibrium dialysis. In the absence of calmodulin, binding of the ATP analogue to this truncated protein was severely impaired, which explains, at least in part, the absolute requirement for calmodulin for the catalytic activity of B. anthracis adenylate cyclase.

Adenosine Triphosphate↗
PubMed2002

N-terminus of the photosystem II manganese stabilizing protein: effects of sequence elongation and truncation.

The importance of the N-terminal domain of manganese stabilizing protein in binding to photosystem II has been previously demonstrated [Eaton-Rye and Murata (1989) Biochim. Biophys. Acta 977, 219-226; Odom and Bricker (1992) Biochemistry 31, 5616-5620]. In this paper, we report results from a systematic study of functional and structural consequences of N-terminal elongation and truncation of manganese stabilizing protein. Precursor manganese stabilizing protein is the unprocessed wild-type protein, which carries an N-terminal extension of 84 amino acids in the form of its chloroplastic signal peptide. Despite its increased size, this protein is able to reconstitute O(2) evolution activity to levels observed with the mature, processed protein, but it also binds nonspecifically to PSII. Truncation of wild-type manganese stabilizing protein by site-directed mutagenesis to remove three N-terminal amino acids, resulting in a mutant called DeltaG3M, causes no loss of activity reconstitution, but this protein also exhibits nonspecific binding. Further truncation of the wild-type protein by ten N-terminal amino acids, producing DeltaE10M, limits binding of manganese stabilizing protein to 1 mol/mol of photosystem II and decreases activity reconstitution to about 65% of that obtained with the wild-type protein. Because two copies of wild type normally bind to photosystem II, amino acids in the domain (4)K-(10)E must be involved in the binding of one copy of manganese stabilizing protein to photosystem II. Spectroscopic analysis (CD and UV spectra) reveals that N-terminal elongation and deletion of manganese stabilizing protein influence its overall conformation, even though secondary structure content is not perturbed. Our data suggest that the solution structure of manganese stabilizing protein attains a more compact solution structure upon removal of N-terminal amino acids.

Amino Acid Sequence↗
PubMed2002

Specificity of the interaction between the Paracoccus denitrificans oxidase and its substrate cytochrome c: comparing the mitochondrial to the homologous bacterial cytochrome c(552), and its truncated and site-directed mutants.

Under in vitro conditions, bacterial cytochrome c oxidases may accept several nonhomologous c-type electron donors, including the evolutionarily related mitochondrial cytochrome c. Several lines of evidence suggest that in intact membranes the heme aa(3) oxidase from Paracoccus denitrificans receives its electrons from the membrane-bound cytochrome c(552). Both the structures of the oxidase and of a heterologously expressed, soluble fragment of the c(552) have been determined recently, but no direct structural information about a static cocomplex is available. Here, we analyze the kinetic properties of the isolated oxidase with the full-size c(552), with two truncated soluble forms, and with a set of site-specific mutants within the presumed docking site of the cytochrome, all heterologously expressed in Escherichia coli. Our data indicate that all three forms, the wild type and both truncations, are fully competent kinetically and exhibit biphasic kinetic behavior, however, under widely different ionic strength conditions. When mutations in lysine residues clustered around the interaction domain were introduced into the smallest fragment of c(552), both kinetic parameters, K(M) and k(cat), were drastically influenced. On the other hand, when the nonmutated truncated form was used to donate electrons to a set of oxidase mutants with replacements clustered along the docking site on subunit II, we observe distinct differences when comparing the kinetic properties of the widely used horse heart cytochrome c with those of the bacterial c(552). We conclude that the specific docking sites for the two types of cytochromes differ to some extent.

Cytochrome c Group↗