scholarly journals Subcloning and characterization of the binding domain of fragment B of diphtheria toxin

1993 ◽  
Vol 294 (3) ◽  
pp. 663-666 ◽  
Author(s):  
Q Y Esbensen ◽  
P O Falnes ◽  
S Olsnes ◽  
I H Madshus
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Diphtheria Toxin ◽  
Binding Domain ◽  
Full Length ◽  
Acidic Ph ◽  
High Affinity ◽  
Ph Values ◽  
Triton X

The binding domain (R domain) of diphtheria toxin as defined from the recently published crystal structure [Choe, Bennett, Fujii, Curmi, Kantardjieff, Collier and Eisenberg (1992) Nature (London) 357, 216-222] was subcloned. The 17 kDa peptide containing amino acids 378-535 from fragment B of diphtheria toxin preceded by the tripeptide Met-His-Gly bound specifically and with high affinity to diphtheria-toxin receptors. It efficiently inhibited the toxicity of full-length toxin. The binding domain entered the detergent phase of Triton X-114 at pH values below 6, indicating that it exposed hydrophobic regions at acidic pH.

scholarly journals Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1208 ◽  
Author(s):  
Mohamed H. Habib ◽  
Henriëtte J. Rozeboom ◽  
Marco W. Fraaije
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Active Site ◽  
Biochemical Characterization ◽  
Sequence Motif ◽  
Acidic Ph ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Ph Values

DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

Cloning and characterization of a human brain Na + -independent transporter for small neutral amino acids that transports d -serine with high affinity

2000 ◽  
Vol 287 (3) ◽  
pp. 231-235 ◽  
Author(s):  
Jun Nakauchi ◽  
Hirotaka Matsuo ◽  
Do Kyung Kim ◽  
Akiteru Goto ◽  
Arthit Chairoungdua ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Brain ◽  
Neutral Amino Acids ◽  
High Affinity

scholarly journals Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

2013 ◽  
Vol 454 (2) ◽  
pp. 311-321 ◽  
Author(s):  
Steven M. Sine ◽  
Sun Huang ◽  
Shu-Xing Li ◽  
Corrie J. B. daCosta ◽  
Lin Chen
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Nicotinic Receptors ◽  
Radioligand Binding ◽  
Binding Domain ◽  
Tyrosine Residue ◽  
Selective Binding ◽  
Conserved Residues ◽  
High Affinity ◽  
Subtype Selective

On the basis of the crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-bungarotoxin, mutagenesis and radioligand-binding measurements show that high-affinity target-selective binding depends on interactions between a single conserved tyrosine residue in α7 and nearby conserved and non-conserved residues.

scholarly journals Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

1995 ◽  
Vol 15 (12) ◽  
pp. 6663-6669 ◽  
Author(s):  
L Trieschmann ◽  
Y V Postnikov ◽  
A Rickers ◽  
M Bustin
Keyword(s):  
Amino Acids ◽  
Structural Features ◽  
Binding Domain ◽  
Full Length ◽  
Modular Structure ◽  
Chromosomal Proteins ◽  
Nucleosome Core ◽  
Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

Receptor-binding domain of human α2-macroglobulin Expression, folding and biochemical characterization of a high-affinity recombinant derivative

FEBS Letters ◽  
1994 ◽  
Vol 344 (2-3) ◽  
pp. 242-246 ◽  
Author(s):  
Thor Las Holtet ◽  
Kåre Lehmann Nielsen ◽  
Michael Etzerodt ◽  
Søren Kragh Moestrup ◽  
Jørgen Gliemann ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Biochemical Characterization ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
High Affinity ◽  
Α2 Macroglobulin

scholarly journals Characterization of thermostable aminoacylase from Geobacillus sp. strain SZN

2019 ◽  
pp. 1-9
Author(s):  
Suzana Adenan ◽  
Chee Fah Wong ◽  
Haniza Hanim Mohd Zain ◽  
Saripah Salbiah Syed Abdul Azziz ◽  
Raja Noor Zaliha Raja Abd. Rahman
Keyword(s):  
Amino Acids ◽  
High Temperatures ◽  
Metal Binding ◽  
Helical Structure ◽  
Thermostable Enzyme ◽  
Point Of View ◽  
Divalent Metal Ions ◽  
Α Helix ◽  
Triton X

Aminoacylase (EC 3.5.1.14) hydrolyzes N–acetylated amino acids to produce amino acids. Although thermostable aminoacylase has been commercially produced since 2004, there was a knowledge gap in the field of understanding aminoacylase thermostability from a structural point of view. This study investigated the physical and structural properties of the purified thermostable aminoacylase SZN. The spectropolarimetry data for structural determination has indicated a gradual decrease of α-helix from 36 to 27.6%, followed by tremendous disorientation of the structure at the transition of temperatures from 60 to 70°C (27.6 to 19.5%). In contrast, the percentage of β-sheet has increased steadily over the tested temperatures. The α-helix, where notable metal binding and catalytic residues are located, was totally weakened at temperatures above 70C, thus resulted in loss of activity. The loss of the α-helical structure could further explain drastic deterioration of activity at temperatures beyond 70C. The activity of aminoacylase SZN was enhanced by divalent metal ions, such as Mn2+ and Cu2+, and inhibited by detergent Triton-X-100. As a conclusion, the isolated aminoacylase SZN was characterized as a thermostable enzyme based on the α-helical structure integrity and functional stability in high temperatures. This enzyme could be used as an alternative enzyme for bioindustries in view of its activity enhancement in high temperatures and stability in various tested inhibitors.

SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF THYROID MEMBRANE TSH BINDING PROTEINS

1979 ◽  
Vol 92 (3) ◽  
pp. 512-521 ◽  
Author(s):  
B. Czarnocka ◽  
J. Nauman ◽  
G. Adler ◽  
W. Kiełczyński
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Gel Filtration ◽  
Soluble Proteins ◽  
The Other ◽  
High Affinity ◽  
Triton X ◽  
Maximal Capacity

ABSTRACT Crude plasma membranes obtained from bovine thyroids were found to possess one class of high affinity, low capacity binding sites for TSH with average association constant (Ka) of 1.301 × 109 m−1 and maximal capacity 8.76 × 10−10 m/mg of protein. Treatment of crude membranes fraction with 0.1 % Triton X-100 and the subsequent sonication in ultrasonic disintegrator resulted in solubilization of membranes proteins with mean recovery of 40.0 ± 6.2 %. Soluble proteins retained the property to bind [125I]TSH, but the binding of the hormone was decreased. The removal of the detergent from the solubilizate by gel filtration on Sephadex LH-20 increased the binding of TSH well above that demonstrated for crude thyroid membranes. The chromatography of soluble proteins on Ultrogel AcA-44 revealed the presence of two TSH binding proteins, one with the molecular weight (m.w.) above 130 000 daltons and the other with the m.w. approximately 30 000 daltons. The electrofocusing of solubilizate on Ampholine resulted in two protein peaks, one at pH 4.0–4.1 and the other at pH 4.4–4.6. The latter peak was shown to bind [125I]TSH specifically. The present results have confirmed the heterogeneous character of solubilized TSH receptor preparation and have shown that the hormone binding sites belong to acid proteins.

scholarly journals Characterization of a Di-leucine–based Signal in the Cytoplasmic Tail of the Nucleotide-pyrophosphatase NPP1 That Mediates Basolateral Targeting but not Endocytosis

2001 ◽  
Vol 12 (10) ◽  
pp. 3004-3015 ◽  
Author(s):  
Valérie Bello ◽  
James W. Goding ◽  
Vicki Greengrass ◽  
Adnan Sali ◽  
Valentina Dubljevic ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Tail ◽  
Full Length ◽  
Apical Surface ◽  
Short Sequence ◽  
Conserved Sequence ◽  
Nucleotide Pyrophosphatase ◽  
Targeting Signal ◽  
Basolateral Targeting

Enzymes of the nucleotide pyrophosphatase/phosphodiesterase (NPPase) family are expressed at opposite surfaces in polarized epithelial cells. We investigated the targeting signal of NPP1, which is exclusively expressed at the basolateral surface. Full-length NPP1 and different constructs and mutants were transfected into the polarized MDCK cell line. Expression of the proteins was analyzed by confocal microscopy and surface biotinylation. The basolateral signal of NPP1 was identified as a di-leucine motif located in the cytoplasmic tail. Mutation of either or both leucines largely redirected NPP1 to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP redirected the apical nucleotide pyrophosphatase/phosphodiesterase NPP3 to the basolateral surface. Full-length NPP1 was not significantly internalized. However, when the cytoplasmic tail was deleted upstream the di-leucine motif or when the six upstream flanking amino acids were deleted, the protein was mainly found intracellularly. Endocytosis experiments indicated that these mutants were endocytosed from the basolateral surface. These results identify the basolateral signal of NPP1 as a short sequence including a di-leucine motif that is dominant over apical determinants and point to the importance of surrounding amino acids in determining whether the signal will function as a basolateral signal only or as an endocytotic signal as well.

Thermodynamic and Kinetic Characterization of Duplex Formation between 2′-O, 4′-C-Methylene-modified Oligoribonucleotides, DNA and RNA

2007 ◽  
Vol 27 (6) ◽  
pp. 327-333 ◽  
Author(s):  
Ulla Christensen
Keyword(s):  
Nucleic Acid ◽  
Full Length ◽  
Locked Nucleic Acid ◽  
Complementary Dna ◽  
High Affinity ◽  
Thermodynamics And Kinetics ◽  
Dna And Rna ◽  
Reaction Thermodynamics ◽  
Duplex Formation

2′-O,4′-C-methylene-linked ribonucleotide derivatives, named LNA (locked nucleic acid) and BNA (bridged nucleic acid) are nucleic acid analogoues that have shown high-affinity recognition of DNA and RNA, and the employment of LNA oligomers for antisense activity, gene regulation and nucleic acid diagnostics seems promising. Here we show kinetic and thermodynamic results on the interaction of a series of 10 bases long LNA–DNA mixmers, gabmers as well as full length LNA's with the complementary DNA, RNA and LNA oligonucleotides in the presence and absence of 10 mM Mg2+- ions. Our results show no significant differences in the reaction thermodynamics and kinetics between the LNA species, only a tendency to stronger duplex formation with the gabmer and mixmer. Introduction of a few LNA's thus may be a better strategy, than using full length LNA's to obtain an oligonucleotide that markedly increases the strength of duplexes formed with the complementary DNA and RNA.

scholarly journals Purification and characterization of antibacterial proteins from granular hemocytes of Indian mud crab, Scylla serrata.

2009 ◽  
Vol 56 (1) ◽  
Author(s):  
Roshan Dinesh Yedery ◽  
Kudumula Venkata Rami Reddy
Keyword(s):  
Amino Acids ◽  
Ejaculatory Duct ◽  
Full Length ◽  
Scylla Serrata ◽  
Antimicrobial Protein ◽  
Microbial Infection ◽  
Rt Pcr ◽  
Pathogen Invasion ◽  
Mangrove Crab

Marine invertebrates depend upon antimicrobial peptides (AMPs) as a major component of innate immunity, as they are rapidly synthesized and diffuse upon pathogen invasion. In this study, we report the identification and characterization of a 11 kDa antimicrobial protein, which we name SSAP (for Scylla serrata antimicrobial protein), from granular hemocytes of the mangrove crab S. serrata. The protein is highly similar to scygonadin, a male-specific AMP isolated from the ejaculatory duct of S. serrata. SSAP was isolated using various chromatographic techniques, viz. ion-exchange, ultra filtration and RP-HPLC, and demonstrated antibacterial activity against Gram positive and Gram negative bacteria. Full length mRNA encoding SSAP was amplified using a combination of RT-PCR and RACE. The nucleotide sequence revealed a full-length ORF of 381 bp coding for a preprotein of 126 amino acids comprising a signal peptide of 24 amino acids and a mature protein of 102 amino acids with a predicted mass of 11435 Da and pI of 5.70. Unlike scygonadin, SSAP is expressed in several tissues of both male and female crabs, as evidenced by RT-PCR, Northern and Western blot analyses. The study suggests that SSAP might be an isoform or a variant of scygonadin and might play an important role in regulating the immunity of the crab upon microbial infection.

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