scholarly journals Mutual conformational changes of tryptophanyl-tRNA synthetase and tRNATrp in the course of their specific interaction

1983 ◽  
Vol 136 (3) ◽  
pp. 559-570 ◽  
Author(s):  
Sergey BERESTEN ◽  
Vladimir SCHEINKER ◽  
Olga FAVOROVA ◽  
Lev KISSELEV
Keyword(s):  
Conformational Changes ◽  
Specific Interaction ◽  
Trna Synthetase

Interaction Of Fibrin And Tissue Plasminogen Activator

1981 ◽  
Author(s):  
P Wallén ◽  
M Rånby
Keyword(s):  
Dissociation Constant ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Conformational Changes ◽  
Specific Interaction ◽  
Kinetic Studies ◽  
Test System ◽  
Activation Rate ◽  
Tissue Plasminogen ◽  
Analytical System

Fibrin itself has a marked influence on fibrinolysis induced by tissue plasminogen activator (TA) indicating a specific interaction. The interaction between fibrin and TA is manifested in two ways, 1) a marked stimulating effect of fibrin on the activation of plasminogen; 2) physical ad- sorbtion of TA on fibrin. By measurement in a sensitive analytical system in which the generation of plasmin is followed by a chromogenic substrate it has been shown that TA is a rather poor activator of plasminogen. In the presence of fibrin the kinetics of the activation is dramatically changed. A stimulation up to 1000-fold is obtained at low plasminogen concentrations. As for activation of native plasminogen (Glu-plasminogen) there is a decrease of km (about 15-fold) as well as an increase of kc (about 80-fold). Fibrinogen has comparatively little effect on the activation rate (at the most 10-fold). The amidolytic activity of TA, using an activator sensitive substrate, is not influenced by fibrin indicating that the effect is not due to conformational changes in the active site region of TA.By varying the concentration of fibrin in the test system it has been demonstrated that the stimulation effect increases suddenly at a fibrin concentration of about 0.01μM. It was suggested that this value represents the dissociation constant of the TA-fibrin complex. However, the amount of fibrin necessary for the adsorbtion of TA in purification experiments indicates a significantly higher dissociation constant (about 0.4 μM). An important difference between the purification experiments and the studies on fibrin stimulation is that plasminogen (plasmin) is absent in the former studies. The formation of a triple complex between fibrin, plasminogen and TA may be the explanation for a more efficient binding of TA in the kinetic studies.

scholarly journals ISOLATION AND STRUCTURE ANALYSIS OF THE MUTANT FORM OF N-TERMINAL CATALYTIC MODULE OF BOS TAURUS TYROSYL-tRNA SYNTHETASE WITH REPLACEMENT OF Trp 40 AND Trp 87 BY ALANINE

2021 ◽  
pp. 27-39
Author(s):  
V. N. Zayets ◽  
L. A. Kolomiiets ◽  
О. Yu. Tsuvarev ◽  
A. I. Kornelyuk
Keyword(s):  
Fluorescence Spectroscopy ◽  
Functional Properties ◽  
Conformational Changes ◽  
Bos Taurus ◽  
Trna Synthetase ◽  
Coli Strain ◽  
Mutant Form ◽  
Compact Structure ◽  
Structural Dynamic ◽  
Cloned Cdna

Aim. Isolation and analysis of the structure of the mutant monotryptophan protein mini BtTyrRS for study of conformational changes of the enzyme at the stage of interaction with tRNA using fluorescence spectroscopy and determination of the effect of tryptophan residues in position 40 and 87 in its structure on the functional properties of the enzyme. Methods. Electrophoresis, metal-chelating affinity chromatography, fluorescence spectroscopy, spatial structure modeling. Results. It was found that the replacement of two codons of Trp by codons of Ala in the cloned cDNA mini BtTyrRS does not affect the synthesis of the mutant form of the enzyme in E. coli strain BL21 (DE3) pLysE. The yield of affinity purified protein on Ni-NTA agarose is on average 3.5 mg per 100 ml of culture medium. Computer modeling of the structure and fluorescence spectroscopy of the monotryptophan form of mini BtTyrRS indicates a compact structure of the mutant enzyme, in which Trp 283 is in an immobilized microenvironment. Conclusions. Affinity purified on Ni-NTA agarose mutant monotryptophan protein mini TyrRS have been obtained which is suitable for fluorescent studies of structural-dynamic and functional properties of the enzyme.

scholarly journals Hairpin RNA-induced conformational change of a eukaryotic-specific lysyl-tRNA synthetase extension and role of adjacent anticodon-binding domain

2020 ◽  
Vol 295 (34) ◽  
pp. 12071-12085
Author(s):  
Sheng Liu ◽  
Maryanne Refaei ◽  
Shuohui Liu ◽  
Aaron Decker ◽  
Jennifer M. Hinerman ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Transition ◽  
Critical Role ◽  
Helical Structure ◽  
Trna Synthetase ◽  
Binding Domain ◽  
Covalent Attachment ◽  
Intrinsically Disordered ◽  
High Affinity ◽  
Rna Hairpins

Human lysyl-tRNA synthetase (hLysRS) is essential for aminoacylation of tRNALys. Higher eukaryotic LysRSs possess an N-terminal extension (Nterm) previously shown to facilitate high-affinity tRNA binding and aminoacylation. This eukaryote-specific appended domain also plays a critical role in hLysRS nuclear localization, thus facilitating noncanonical functions of hLysRS. The structure is intrinsically disordered and therefore remains poorly characterized. Findings of previous studies are consistent with the Nterm domain undergoing a conformational transition to an ordered structure upon nucleic acid binding. In this study, we used NMR to investigate how the type of RNA, as well as the presence of the adjacent anticodon-binding domain (ACB), influences the Nterm conformation. To explore the latter, we used sortase A ligation to produce a segmentally labeled tandem-domain protein, Nterm–ACB. In the absence of RNA, Nterm remained disordered regardless of ACB attachment. Both alone and when attached to ACB, Nterm structure remained unaffected by titration with single-stranded RNAs. The central region of the Nterm domain adopted α-helical structure upon titration of Nterm and Nterm–ACB with RNA hairpins containing double-stranded regions. Nterm binding to the RNA hairpins resulted in CD spectral shifts consistent with an induced helical structure. NMR and fluorescence anisotropy revealed that Nterm binding to hairpin RNAs is weak but that the binding affinity increases significantly upon covalent attachment to ACB. We conclude that the ACB domain facilitates induced-fit conformational changes and confers high-affinity RNA hairpin binding, which may be advantageous for functional interactions of LysRS with a variety of different binding partners.

scholarly journals CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence

2008 ◽  
Vol 205 (3) ◽  
pp. 725-735 ◽  
Author(s):  
Emma J. Petrie ◽  
Craig S. Clements ◽  
Jie Lin ◽  
Lucy C. Sullivan ◽  
Darryl Johnson ◽  
...  
Keyword(s):  
Human Leukocyte Antigen ◽  
Conformational Changes ◽  
Specific Interaction ◽  
Human Leukocyte ◽  
Hla Class I ◽  
Binding Mode ◽  
Leader Sequence ◽  
Structural Basis ◽  
Peptide Ligand ◽  
Leukocyte Antigen

The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A–HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a “lock and key” interaction is typical of innate receptor–ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.

Structural Studies of Lysyl-tRNA Synthetase:  Conformational Changes Induced by Substrate Binding†

Biochemistry ◽  
2000 ◽  
Vol 39 (42) ◽  
pp. 12853-12861 ◽  
Author(s):  
Silvia Onesti ◽  
Gianluigi Desogus ◽  
Annie Brevet ◽  
Josiane Chen ◽  
Pierre Plateau ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Substrate Binding ◽  
Trna Synthetase ◽  
Structural Studies

Interactions of yeast valyl-tRNA synthetase with RNAs and conformational changes of the enzyme

1979 ◽  
Vol 129 (3) ◽  
pp. 483-500 ◽  
Author(s):  
Giuseppe Zaccaï ◽  
Philippe Morin ◽  
Bernard Jacrot ◽  
Dino Moras ◽  
Jean-Claude Thierry ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Trna Synthetase

scholarly journals Development of an Impedimetric Aptasensor for Label Free Detection of Patulin in Apple Juice

Molecules ◽  
2019 ◽  
Vol 24 (6) ◽  
pp. 1017 ◽  
Author(s):  
Reem Khan ◽  
Sondes Ben Aissa ◽  
Tauqir Sherazi ◽  
Gaelle Catanante ◽  
Akhtar Hayat ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Apple Juice ◽  
Electrochemical Impedance ◽  
Limit Of Detection ◽  
Diazonium Salt ◽  
Specific Interaction ◽  
Quantitative Detection ◽  
Label Free ◽  
Analytical Technique ◽  
Label Free Detection

In the present work, an aptasensing platform was developed for the detection of a carcinogenic mycotoxin termed patulin (PAT) using a label-free approach. The detection was mainly based on a specific interaction of an aptamer immobilized on carbon-based electrode. A long linear spacer of carboxy-amine polyethylene glycol chain (PEG) was chemically grafted on screen-printed carbon electrodes (SPCEs) via diazonium salt in the aptasensor design. The NH2-modified aptamer was then attached covalently to carboxylic acid groups of previously immobilized bifunctional PEG to build a diblock macromolecule. The immobilized diblocked molecules resulted in the formation of long tunnels on a carbon interface, while the aptamer was assumed as the gate of these tunnels. Upon target analyte binding, the gates were assumed to be closed due to conformational changes in the structure of the aptamer, increasing the resistance to the charge transfer. This increase in resistance was measured by electrochemical impedance spectroscopy, the main analytical technique for the quantitative detection of PAT. Encouragingly, a good linear range between 1 and 25 ng was obtained. The limit of detection and limit of quantification was 2.8 ng L−1 and 4.0 ng L−1, respectively. Selectivity of the aptasensor was confirmed with mycotoxins commonly occurring in food. The developed apta-assay was also applied to a real sample, i.e., fresh apple juice spiked with PAT, and toxin recovery up to 99% was observed. The results obtained validated the suitability and selectivity of the developed apta-assay for the identification and quantification of PAT in real food samples.

scholarly journals Intracellular Ca2+ regulation of H+/Ca2+ antiporter YfkE mediated by a Ca2+ mini-sensor

2020 ◽  
Vol 117 (19) ◽  
pp. 10313-10321
Author(s):  
Shuo Lu ◽  
Zhenlong Li ◽  
Alemayehu A. Gorfe ◽  
Lei Zheng
Keyword(s):  
Conformational Changes ◽  
Regulatory Mechanism ◽  
Membrane Surface ◽  
Specific Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dynamics Simulation ◽  
Adjacent Segment ◽  
Inhibitory Peptide ◽  
Translocation Pathway

The H+/Ca2+ (calcium ion) antiporter (CAX) plays an important role in maintaining cellular Ca2+ homeostasis in bacteria, yeast, and plants by promoting Ca2+ efflux across the cell membranes. However, how CAX facilitates Ca2+ balance in response to dynamic cytosolic Ca2+ perturbations is unknown. Here, we identified a type of Ca2+ “mini-sensor” in YfkE, a bacterial CAX homolog from Bacillus subtilis. The mini-sensor is formed by six tandem carboxylate residues within the transmembrane (TM)5-6 loop on the intracellular membrane surface. Ca2+ binding to the mini-sensor triggers the transition of the transport mode of YfkE from a high-affinity to a low-affinity state. Molecular dynamics simulation and fluorescence resonance energy transfer analysis suggest that Ca2+ binding to the mini-sensor causes an adjacent segment, namely, the exchanger inhibitory peptide (XIP), to move toward the Ca2+ translocation pathway to interact with TM2a in an inward-open cavity. The specific interaction was demonstrated with a synthetic peptide of the XIP, which inhibits YfkE transport and interrupts conformational changes mediated by the mini-sensor. By comparing the apo and Ca2+-bound CAX structures, we propose the following Ca2+ transport regulatory mechanism of YfkE: Ca2+ binding to the mini-sensor induces allosteric conformational changes in the Ca2+ translocation pathway via the XIP, resulting in a rearrangement of the Ca2+-binding transport site in the midmembrane. Since the Ca2+ mini-sensor and XIP sequences are also identified in other CAX homologs and/or Ca2+ transporters, including the mammalian Na+/Ca2+ exchanger (NCX), our study provides a regulatory mechanism for the Ca2+/cation transporter superfamily.

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