scholarly journals Primary sequence and domain structure of chicken vinculin

1989 ◽  
Vol 259 (2) ◽  
pp. 453-461 ◽  
Author(s):  
G J Price ◽  
P Jones ◽  
M D Davison ◽  
B Patel ◽  
R Bendori ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Domain Structure ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Phosphorylation Site ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Untranslated Sequence ◽  
Globular Head

We have determined the complete sequence of chick vinculin from two overlapping cDNA clones. The vinculin mRNA consists of 262 bp of 5' untranslated sequence, an open reading frame of 3195 bp (excluding the initiation codon) and a long 3' untranslated sequence (greater than 2 kb). Chick vinculin contains 1066 amino acid residues, and has a deduced molecular mass of 116,933 Da. Analysis of the domain structure of vinculin shows that the molecule can be cleaved by V8 proteinase into a 90 kDa globular head and a 32 kDa tail region, the latter of which could further be cleaved into a 27 kDa polypeptide. The 90 kDa globular head contains the N-terminus of vinculin, three 112-residue repeats (residues 259-589), and extends to approximately residue 850. Gel overlay experiments show that it also contains a binding site for the cytoskeletal protein talin. The talin-binding domain was further localized to the N-terminal 398 amino acid residues of the protein by expression in vitro of this region from a vinculin cDNA cloned into the Bluescript SK+ vector. The head and tail domains are apparently separated by a proline-rich region that contains V8-proteinase-cleavage sites and a candidate tyrosine (822)-phosphorylation site. Secondary-structure prediction suggests that the head and tail domains contain alpha-helical regions separated by short stretches of turn/coil. Comparison of the chick with a partial human sequence reveals that vinculin is a highly conserved protein. In chickens Southern-blot analysis is consistent with a single vinculin gene, and it is therefore likely that vinculin, and its higher-molecular-mass isoform termed metavinculin, arise through alternative splicing.

HATODAS II – heavy-atom database system with potentiality scoring

2009 ◽  
Vol 42 (3) ◽  
pp. 540-544 ◽  
Author(s):  
Michihiro Sugahara ◽  
Yukuhiko Asada ◽  
Hiroki Shimada ◽  
Hideyuki Taka ◽  
Naoki Kunishima
Keyword(s):  
Amino Acid ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Solvent Accessibility ◽  
Heavy Atom ◽  
Database System ◽  
Protein Crystals ◽  
Amino Acid Residues ◽  
Sequence Alignments ◽  
Homologous Proteins

HATODAS II is the second version of HATODAS (the Heavy-Atom Database System), which suggests potential heavy-atom reagents for the derivatization of protein crystals. The present expanded database contains 3103 heavy-atom binding sites, which is four times more than the previous version. HATODAS II has three new criteria to evaluate the feasibility of the search results: (1) potentiality scoring for the predicted heavy-atom reagents, (2) exclusion of the disordered amino acid residues based on the secondary structure prediction and (3) consideration of the solvent accessibility of amino acid residues from a homology model. In the point mutation option, HATODAS II suggests possible mutation sites into reactive amino acid residues such as Met, Cys and His, on the basis of multiple sequence alignments of homologous proteins. These new features allow the user to make a well informed decision as to the possible heavy-atom derivatization experiments of protein crystals.

Cloning and Characterization of Three Genes Encoding LMW-GSs from Triticum aestivum, Cultival Cheyenne

2012 ◽  
Vol 554-556 ◽  
pp. 1116-1120 ◽  
Author(s):  
Mei Rong Chen ◽  
Xing Shen ◽  
Lin Li ◽  
Song Qing Hu
Keyword(s):  
Amino Acid ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Amino Acid Residues ◽  
Repetitive Domain ◽  
Genebank Accession ◽  
Genes Encoding ◽  
Α Helix ◽  
Β Sheet

Three low molecular weight subunit genes, named LMW-CND1 (GeneBank accession JQ780048), LMW-CND2 (GeneBank accession JQ779840), LMW-CND3 (GeneBank accession JQ779841), with a ORF of 1053 bp, 903 bp, 969 bp, respectively, were isolated from cv. Cheyenne and characterized detailed in molecular level. The proteins encoded by the genes, with 350, 300, 322 amino acid residues respectively, differ only in repetitive domain of sequences due to insertion or deletion of repeats in this domain. Highly similarity in amino-acid sequence between these three subunits and other published LMW-GSs was also observed, showing that all three genes published here are typical LMW-GS genes and closely related to the genes on chromosome 1D. Besides, secondary structure prediction of proteins indicated that, in the three LMW-GSs, random loop accounts for no less than 70 %, α-helix amounts to 26 %, average, and only 1.4 %~1.7 % is β-sheet.

scholarly journals Molecular and Mechanistic Characterization of PddB, the First PLP-Independent 2,4-Diaminobutyric Acid Racemase Discovered in an Actinobacterial D-Amino Acid Homopolymer Biosynthesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Kazuya Yamanaka ◽  
Ryo Ozaki ◽  
Yoshimitsu Hamano ◽  
Tadao Oikawa
Keyword(s):  
Amino Acid ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Sequence Similarity ◽  
Structural Difference ◽  
Structural Modeling ◽  
Specific Substrate ◽  
Amino Acid Residues ◽  
Diaminobutyric Acid

We recently disclosed that the biosynthesis of antiviral γ-poly-D-2,4-diaminobutyric acid (poly-D-Dab) in Streptoalloteichus hindustanus involves an unprecedented cofactor independent stereoinversion of Dab catalyzed by PddB, which shows weak homology to diaminopimelate epimerase (DapF). Enzymological properties and mechanistic details of this enzyme, however, had remained to be elucidated. Here, through a series of biochemical characterizations, structural modeling, and site-directed mutageneses, we fully illustrate the first Dab-specific PLP-independent racemase PddB and further provide an insight into its evolution. The activity of the recombinant PddB was shown to be optimal around pH 8.5, and its other fundamental properties resembled those of typical PLP-independent racemases/epimerases. The enzyme catalyzed Dab specific stereoinversion with a calculated equilibrium constant of nearly unity, demonstrating that the reaction catalyzed by PddB is indeed racemization. Its activity was inhibited upon incubation with sulfhydryl reagents, and the site-directed substitution of two putative catalytic Cys residues led to the abolishment of the activity. These observations provided critical evidence that PddB employs the thiolate-thiol pair to catalyze interconversion of Dab isomers. Despite the low levels of sequence similarity, a phylogenetic analysis of PddB indicated its particular relevance to DapF among PLP-independent racemases/epimerases. Secondary structure prediction and 3D structural modeling of PddB revealed its remarkable conformational analogy to DapF, which in turn allowed us to predict amino acid residues potentially responsible for the discrimination of structural difference between diaminopimelate and its specific substrate, Dab. Further, PddB homologs which seemed to be narrowly distributed only in actinobacterial kingdom were constantly encoded adjacent to the putative poly-D-Dab synthetase gene. These observations strongly suggested that PddB could have evolved from the primary metabolic DapF in order to organize the biosynthesis pathway for the particular secondary metabolite, poly-D-Dab. The present study is on the first molecular characterization of PLP-independent Dab racemase and provides insights that could contribute to further discovery of unprecedented PLP-independent racemases.

scholarly journals Isolation and sequence analysis of a cDNA encoding rat liver α-l-fucosidase

1989 ◽  
Vol 264 (3) ◽  
pp. 695-701 ◽  
Author(s):  
K J Fisher ◽  
N N Aronson
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Signal Peptide ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Expression Library ◽  
Cdna Insert ◽  
Untranslated Sequence ◽  
N Terminus

cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5′ untranslated sequence, 78 nucleotide residues of 3′ untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].

scholarly journals Molecular cloning of two human liver 3 α-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are identical with chlordecone reductase and bile-acid binder

1994 ◽  
Vol 299 (2) ◽  
pp. 545-552 ◽  
Author(s):  
Y Deyashiki ◽  
A Ogasawara ◽  
T Nakayama ◽  
M Nakanishi ◽  
Y Miyabe ◽  
...  
Keyword(s):  
Bile Acid ◽  
Amino Acid ◽  
Human Liver ◽  
Polyclonal Antibodies ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Human Bile ◽  
Coding Regions ◽  
Computer Based

Human liver contains two dihydrodiol dehydrogenases, DD2 and DD4, associated with 3 alpha-hydroxysteroid dehydrogenase activity. We have raised polyclonal antibodies that cross-reacted with the two enzymes and isolated two 1.2 kb cDNA clones (C9 and C11) for the two enzymes from a human liver cDNA library using the antibodies. The clones of C9 and C11 contained coding sequences corresponding to 306 and 321 amino acid residues respectively, but lacked 5′-coding regions around the initiation codon. Sequence analyses of several peptides obtained by enzymic and chemical cleavages of the two purified enzymes verified that the C9 and C11 clones encoded DD2 and DD4 respectively, and further indicated that the sequence of DD2 had at least additional 16 residues upward from the N-terminal sequence deduced from the cDNA. There was 82% amino acid sequence identity between the two enzymes, indicating that the enzymes are genetic isoenzymes. A computer-based comparison of the cDNAs of the isoenzymes with the DNA sequence database revealed that the nucleotide and amino acid sequences of DD2 and DD4 are virtually identical with those of human bile-acid binder and human chlordecone reductase cDNAs respectively.

scholarly journals Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

2003 ◽  
Vol 50 (1) ◽  
pp. 269-278
Author(s):  
Amr M Shabaan ◽  
Magdy M Mohamed ◽  
Mohga S Abdallah ◽  
Hayat M Ibrahim ◽  
Amr M Karim
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Vaccine Development ◽  
Complete Sequence ◽  
Cdna Clones ◽  
Western Blots ◽  
Calculated Molecular Mass ◽  
Reading Frame

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

scholarly journals The complete amino acid sequence of three alcohol dehydrogenase alleloenzymes (AdhN-11, AdhS and AdhUF) from the fruitfly Drosophila melanogaster

1980 ◽  
Vol 187 (3) ◽  
pp. 875-883 ◽  
Author(s):  
D R Thatcher
Keyword(s):  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Aspartic Acid ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
British Library ◽  
Horse Liver ◽  
And Staphylococcus Aureus

The sequence of three alcohol dehydrogenase alleloenzymes from the fruitfly Drosophila melanogaster has been determined by the sequencing of peptides produced by trypsin, chymotrypsin, thermolysin, pepsin and Staphylococcus aureus-V8-proteinase digestion. The amino acid sequence shows no obvious homology with the published sequences of the horse liver and yeast enzymes, and secondary structure prediction suggests that the nucleotide-binding domain is located in the N-terminal half of the molecule. The amino acid substitutions between AdhN-11 (a point mutation of AdhF), AdhS and AdhUF alleloenzymes were identified. AdhN-11 alcohol dehydrogenase differed from the other two by a glycine-14-(AdhS and AdhUF)-to-aspartic acid substitution, the AdhS enzyme from AdhN-11 and AdhUF enzymes by a threonine-192-(AdhN-11 and AdhUF)-to-lysine (AdhS) substitution and the AdhUF enzyme was found to differ by an alanine-45-(AdhS and AdhN-11)-to-aspartic acid (AdhUF) charge substitution and a ‘silent’ asparagine-8-(AdhS and AdhN-11)-to-alanine (AdhUF) substitution. Detailed sequence evidence has been deposited as Supplementary Publication SUP 50107 (36 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

scholarly journals A Strain of Bacillus thuringiensis Containing a Novel cry7Aa2 Gene that Is Toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)

Insects ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 259 ◽  
Author(s):  
Mikel Domínguez-Arrizabalaga ◽  
Maite Villanueva ◽  
Ana Beatriz Fernandez ◽  
Primitivo Caballero
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Molecular Mass ◽  
Leptinotarsa Decemlineata ◽  
Insect Pests ◽  
Amino Acid Residues ◽  
Cry Protein ◽  
Lc50 Value ◽  
Leptinotarsa Decemlineata Say

The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity compared to Cry7Aa1 and a predicted molecular mass of 129.4 kDa. The primary structure of Cry7Aa2, which had eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that after in vitro trypsin incubation, the protein was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae, one major proteinase-resistant fragment of slightly smaller size was produced. The spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 value of 18.8 μg/mL, which was statistically similar to the estimated LC50 of 20.8 μg/mL for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 3.8-fold to LC50 = 4.9 μg/mL. The potential advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.

Modèle topologique de la structure d’un antiport vacuolaire de type NHX chez la vigne cultivée (Vitis vinifera)

Botany ◽  
2009 ◽  
Vol 87 (3) ◽  
pp. 339-347 ◽  
Author(s):  
Mohsen Hanana ◽  
Olivier Cagnac ◽  
Ahmed Mliki ◽  
Eduardo Blumwald
Keyword(s):  
Amino Acid ◽  
Vitis Vinifera ◽  
Molecular Mass ◽  
Electrostatic Interactions ◽  
Functional Characterization ◽  
Terminal Region ◽  
Vitis Vinifera L ◽  
Amino Acid Residues ◽  
Transmembrane Segments ◽  
Α Helix

After identifying and isolating a grapevine ( Vitis vinifera L.) NHX vacuolar antiporter and before initializing functional genomic studies, we juged necessary to acquire a minimum of knowledge about the VvNHX1 protein. Thus, we realized a bioinformatic analysis to determine its basic characteristics and to get structural informations that could guide us through the functional characterization. We have determined important physico-chemical parameters (molecular mass, isoelectric point, hydrophobic regions, etc.) and obtained interesting structural data (primary, secondary, and tertiary structures; conserved domains and interaction motives; etc.). The VvNHX1 gene, which encodes this 541 amino-acid protein with a predicted molecular mass of 60 kDa, is made of 14 exons and measures 6.5 kb. The amino-acidic composition of this protein is very important, in particular, for the establishment of the α-helix structure, which represents more than 50% of the protein, but also for charge distribution, which generates critical electrostatic interactions for the ionic flux. The secondary structure of VvNHX1 contains multiple transmembrane α-helix segments that are made of hydrophobic amino-acid residues, thus facilitating its insertion in the membrane. Globally, VvNHX1 has one hydrophobic N-terminal region, made of 10 transmembrane segments with 440 amino-acid residues, and one hydrophilic C-terminal region, made of 100 residues. The region located between the fourth and fifth transmembrane segments represents, with its structure mainly helicoidal and the presence of a favourable electrostatic environment, the pore where cation flux is performed across the membrane. VvNHX1 contains various interaction domains as well as several putative posttranslational modification sites, mainly at the C-terminus but also at the N-terminus, that play an important part in regulating protein activities, influence protein structural stability, or interact with other proteins or signalling molecules.

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