A Disulfide Cross-linking Strategy Useful for Studying Ligand-induced Structural Changes in GPCRs

2010 ◽  
pp. 147-167
Author(s):  
Jian Hua Li ◽  
Stuart D. C. Ward ◽  
Sung-Jun Han ◽  
Fadi F. Hamdan ◽  
Jrgen Wess
Keyword(s):  
Structural Changes ◽  
Cross Linking

Embedding Procedure for Water Swollen Samples

1970 ◽  
Vol 28 ◽  
pp. 504-505
Author(s):  
Ann M. Thomas ◽  
Virginia Shemeley
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Ion Exchange ◽  
Structural Changes ◽  
Cross Linking ◽  
Ion Exchange Resins ◽  
Transmission Electron ◽  
First Pass ◽  
Exchange Resins

Those samples which swell rapidly when exposed to water are, at best, difficult to section for transmission electron microscopy. Some materials literally burst out of the embedding block with the first pass by the knife, and even the most rapid cutting cycle produces sections of limited value. Many ion exchange resins swell in water; some undergo irreversible structural changes when dried. We developed our embedding procedure to handle this type of sample, but it should be applicable to many materials that present similar sectioning difficulties.The purpose of our embedding procedure is to build up a cross-linking network throughout the sample, while it is in a water swollen state. Our procedure was suggested to us by the work of Rosenberg, where he mentioned the formation of a tridimensional structure by the polymerization of the GMA biproduct, triglycol dimethacrylate.

Structural Changes in Cotton: Effect of Premercerization Conditions on Subsequent Cross-Linking

1969 ◽  
Vol 39 (2) ◽  
pp. 148-154 ◽  
Author(s):  
Ricardo H. Wade ◽  
Tyrone L. Vigo
Keyword(s):  
Structural Changes ◽  
Cotton Effect ◽  
Cross Linking ◽  
Fragmentation Pattern ◽  
Cellulose I ◽  
Metal Hydroxides ◽  
Normal Length ◽  
Expansion Pattern ◽  
The Cross ◽  
Fiber Fragmentation

Structural changes due to tension applied to caustic-swollen yarns were studied. It was found that the cross-linking of yarns which had been mercerized slack, at normal length, or slack and then restretched to normal length produced differences in tenacity and X-ray orientation. It was concluded that this was due to structural rearrangements induced by the application of load to fiber systems. Differences in wet pickup of the cross-linking resin were shown to produce differences in the fiber fragmentation pattern but not in the layer-expansion pattern. Only a fraction of the added cross-linking resin was considered to have contributed to the properties usually attributed to cross-linked yarns. The regions believed responsible for the effect of cross-linking are the less ordered lattices close to the crystalline structures. The strength retained after cross-linking was dependent on the tension and the method of its application. Differences in the degree of conversion of cellulose I to cellulose II were noted in the slack-mercerized yarns treated with different alkali metal hydroxides. These differences, with the exception of lithium hydroxide, correlated with swelling effectiveness of the alkalis used.

scholarly journals Crosslinked Recombinant-Ara h 1 Catalyzed by Microbial Transglutaminase: Preparation, Structural Characterization and Allergic Assessment

Foods ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1508
Author(s):  
Yang Tian ◽  
Chenglong Liu ◽  
Wentong Xue ◽  
Zhongfu Wang
Keyword(s):  
Disulfide Bonds ◽  
Structural Changes ◽  
Structural Features ◽  
Microbial Transglutaminase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cross Linking ◽  
Intrinsic Structure ◽  
Ara H 1 ◽  
Molecular Forces ◽  
The One

As the one of the major allergens in peanut, the allergenicity of Ara h 1 is influenced by its intrinsic structure, which can be modified by different processing. However, molecular information in this modification has not been clarified to date. Here, we detected the influence of microbial transglutaminase (MTG) catalyzed cross-linking on the recombinant peanut protein Ara h 1 (rAra h 1). Electrophoresis and spectroscopic methods were used to analysis the structural changes. The immunoreactivity alterations were characterized by enzyme linked immunosorbent assay (ELISA), immunoblotting and degranulation test. Structural features of cross-linked rAra h 1 varied at different reaction stages. Hydrogen bonds and disulfide bonds were the main molecular forces in polymers induced by heating and reducing. In MTG-catalyzed cross-linking, ε-(γ-glutamyl) lysine isopeptide bonds were formed, thus inducing a relatively stable structure in polymers. MTG catalyzed cross-linking could modestly but significantly reduce the immunoreactivity of rAra h 1. Decreased content of conserved secondary structures led to a loss of protection of linear epitopes. Besides, the reduced surface hydrophobic index and increased steric hindrance of rAra h 1 made it more difficult to bind with antibodies, thus hindering the subsequent allergic reaction.

G-actin conformational change and polymerization induced by paraquat

1998 ◽  
Vol 76 (4) ◽  
pp. 583-591 ◽  
Author(s):  
Isabella DalleDonne ◽  
Aldo Milzani ◽  
Roberto Colombo
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Structural Changes ◽  
Cell Injury ◽  
Atp Hydrolysis ◽  
Protein Composition ◽  
Dnase I ◽  
Cross Linking ◽  
Cytoskeletal Protein ◽  
Actin Assembly

Paraquat (1,1´-dimethyl-4,4´-bipyridilium dichloride) is a broad-spectrum herbicide that is highly toxic to animals (including man), the major lesion being in the lung. In mammalian cells, paraquat causes deep alterations in the organization of the cytoskeleton, marked decreases in cytoskeletal protein synthesis, and alterations in cytoskeletal protein composition; therefore, the involvement of the cytoskeleton in cell injury by paraquat was suggested. We previously demonstrated that monomeric actin binds paraquat; moreover, prolonged actin exposure to paraquat, in depolymerizing medium, induces the formation of actin aggregates, which are built up by F-actin. In this work we have shown that the addition of paraquat to monomeric actin results in a strong quenching of Trp-79 and Trp-86 fluorescence. Trypsin digestion experiments demonstrated that the sequence 61-69 on actin subdomain 2 undergoes paraquat-dependent conformational changes. These paraquat-induced structural changes render actin unable to completely inhibit DNase I. By using intermolecular cross-linking to characterize oligomeric species formed during paraquat-induced actin assembly, we found that the herbicide causes the formation of actin oligomers characterized by subunit-subunit contacts like those occurring in oligomers induced by polymerizing salts (i.e., between subdomain 1 on one actin subunit and subdomain 4 on the adjacent subunit). Furthermore, the oligomerization of G-actin induced by paraquat is paralleled by ATP hydrolysis.Key words: actin, paraquat, subdomain 2, DNase I, ATP hydrolysis.

scholarly journals Ion Charge Influence on the Molecular Structure of Polyethylene Terephthalate Films after Irradiation with Swift Heavy Ions

Crystals ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 479
Author(s):  
Adil Z. Tuleushev ◽  
Maxim V. Zdorovets ◽  
Artem L. Kozlovskiy ◽  
Fiona E. Harrison
Keyword(s):  
Polyethylene Terephthalate ◽  
Structural Changes ◽  
Diffraction Method ◽  
Dipole Interaction ◽  
Heavy Ion ◽  
Cross Linking ◽  
X Ray Diffraction ◽  
Chain Molecules ◽  
Repeat Units ◽  
Pet Film

We report here experimental results investigating the influence of the initial swift heavy ion charge on the structure of polyethylene terephthalate (PET) film after irradiation, using a structurally sensitive X-ray diffraction method. Kr ions with an energy of 100 MeV and charges of 13+, 14+, and 15+ were each used at irradiation fluences of 5 × 1010, 7.5 × 1010, 1 × 1011, 2.5 × 1011 and 5 × 1011 ions/cm2. At constant energy and irradiation fluence, the post-irradiation structural changes in PET film show a clear dependence on the initial ion charge. As either the fluence or ion charge increase, the latent tracks begin to overlap, leading to cross-linking of PET chain molecules to form rotational isomers (rotamers). We use the fluence corresponding to the onset of overlapping to estimate the size of latent tracks for different ion charges. At the highest fluences, the latent tracks become entirely overlapped, and the interchain cross-linking extends throughout the whole film. Since this cross-linking is due to the dipole–dipole interaction of subunits of repeat units of PET chain molecules, it is reversible, in contrast to the well-known chemical cross-linking of polymer chain molecules under irradiation.

Monitoring Conformational Changes in Protein Complexes Using Chemical Cross-Linking and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: The Effect of Calcium Binding on the Calmodulin—Melittin Complex

2007 ◽  
Vol 13 (4) ◽  
pp. 281-290 ◽  
Author(s):  
Petr Novak ◽  
Vladimir Havlicek ◽  
Peter J. Derrick ◽  
Kyle A. Beran ◽  
Sajid Bashir ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Fourier Transform ◽  
Conformational Changes ◽  
Calcium Binding ◽  
Cyclotron Resonance ◽  
Structural Changes ◽  
Cross Linking ◽  
Ion Cyclotron Resonance ◽  
Ion Cyclotron ◽  
Chemical Cross Linking

Calmodulin is an EF hand calcium binding protein. Its binding affinities to various protein/peptide targets often depend on the conformational changes induced by the binding of calcium. One such target is melittin, which binds tightly to calmodulin in the presence of calcium, and inhibits its function. Chemical cross-linking combined with Fourier transform ion cyclotron resonance mass spectrometry has been employed to investigate the coordination of calmodulin and melittin in the complex at different concentrations of calcium. This methodology can be used to monitor structural changes in proteins induced by ligand binding and to study the effects these changes have on non-covalent interactions between proteins. Cross-linking results indicate that the binding place of the first melittin in the calcium-free calmodulin form is the same as in the calcium-loaded calmodulin/melittin complex.

scholarly journals Increased tissue transglutaminase activity contributes to central vascular stiffness in eNOS knockout mice

2013 ◽  
Vol 305 (6) ◽  
pp. H803-H810 ◽  
Author(s):  
Sung Mee Jung ◽  
Simran Jandu ◽  
Jochen Steppan ◽  
Alexey Belkin ◽  
Steven S. An ◽  
...  
Keyword(s):  
Mean Arterial Pressure ◽  
Arterial Pressure ◽  
Knockout Mice ◽  
Structural Changes ◽  
Tissue Transglutaminase ◽  
Vascular Stiffness ◽  
Cross Linking ◽  
Aortic Tissue ◽  
Dimension Analysis ◽  
And Function

Nitric oxide (NO) can modulate arterial stiffness by regulating both functional and structural changes in the arterial wall. Tissue transglutaminase (TG2) has been shown to contribute to increased central aortic stiffness by catalyzing the cross-linking of matrix proteins. NO S-nitrosylates and constrains TG2 to the cytosolic compartment and thereby holds its cross-linking function latent. In the present study, the role of endothelial NO synthase (eNOS)-derived NO in regulating TG2 function was studied using eNOS knockout mice. Matrix-associated TG2 and TG2 cross-linking function were higher, whereas TG2 S-nitrosylation was lower in the eNOS−/− compared with wild-type (WT) mice. Pulse-wave velocity (PWV) and blood pressure measured noninvasively were elevated in the eNOS−/− compared with WT mice. Intact aortas and decellularized aortic tissue scaffolds of eNOS−/− mice were significantly stiffer, as determined by tensile testing. The carotid arteries of the eNOS−/− mice were also stiffer, as determined by pressure-dimension analysis. Invasive methods to determine the PWV-mean arterial pressure relationship showed that PWV in eNOS−/− and WT diverge at higher mean arterial pressure. Thus eNOS-derived NO regulates TG2 localization and function and contributes to vascular stiffness.

scholarly journals Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling

2020 ◽  
Vol 117 (8) ◽  
pp. 4088-4098 ◽  
Author(s):  
Craig Gutierrez ◽  
Ilan E. Chemmama ◽  
Haibin Mao ◽  
Clinton Yu ◽  
Ignacia Echeverria ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Dynamics ◽  
Structural Changes ◽  
Structural Model ◽  
Protein Complexes ◽  
Cop9 Signalosome ◽  
Cross Linking ◽  
E3 Ligases ◽  
Protein Ubiquitination ◽  
Mass Spectometry

The COP9 signalosome (CSN) is an evolutionarily conserved eight-subunit (CSN1–8) protein complex that controls protein ubiquitination by deneddylating Cullin-RING E3 ligases (CRLs). The activation and function of CSN hinges on its structural dynamics, which has been challenging to decipher by conventional tools. Here, we have developed a multichemistry cross-linking mass spectrometry approach enabled by three mass spectometry-cleavable cross-linkers to generate highly reliable cross-link data. We applied this approach with integrative structure modeling to determine the interaction and structural dynamics of CSN with the recently discovered ninth subunit, CSN9, in solution. Our results determined the localization of CSN9 binding sites and revealed CSN9-dependent structural changes of CSN. Together with biochemical analysis, we propose a structural model in which CSN9 binding triggers CSN to adopt a configuration that facilitates CSN–CRL interactions, thereby augmenting CSN deneddylase activity. Our integrative structure analysis workflow can be generalized to define in-solution architectures of dynamic protein complexes that remain inaccessible to other approaches.

scholarly journals Effect of Elastomeric Nanoparticles on Polystyrene/Organic Nanocomposites

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Sungwon Ma ◽  
Yonathan Thio
Keyword(s):  
Percolation Threshold ◽  
Self Assembly ◽  
Structural Changes ◽  
Volume Concentration ◽  
Filler Loading ◽  
Cross Linking ◽  
Zero Shear Viscosity ◽  
Threshold Theory ◽  
Vulcanization Process ◽  
Nanosized Filler

The rheological behavior of nanosheet composites and the effect of morphology between elastomeric nanofiber and nanosheet composites were studied using a Cross-Williamson model and critical volume concentration was investigated by percolation threshold theory for fiber and sheet morphologies. Nanofiber and nanosheet particles were synthesized by a cold vulcanization process using a S2Cl2cross-linking reagent resulting from self-assembly of a PS-PI block copolymer. Nanofiber and nanosheet characterization was done by SEM. Rheological properties were measured and analyzed in terms of varying nanofiller and nanosheet loading from 0.5 to 10 wt%. For the nanofiber and nanosheet composites, the moduli were increased with increasing filler loading, whereas moduli of SI23 and SI43 composite decreased with increasing content. Both nanofiber and nanosheet composites showed a nanosized filler effect and their structural changes were between 5 and 10 wt%. Cross-Williamson three-parameter model was used to find zero-shear viscosity and relaxation time. Percolation threshold theory was used to study structural changes and calculate values.

Sign in / Sign up

Export Citation Format

Share Document