Linker Complex Research Articles

Synthesis of a Platinum Linker Complex as a Scaffold for the Hybridization of Naturally Occurring DNA and Gold Nanoparticles

Synthesis of a Platinum Linker Complex as a Scaffold for the Hybridization of Naturally Occurring DNA and Gold Nanoparticles

Bromoargentate/g-C3N4 heterojunction by in situ growth: 2-D bromoargentate framework with a transition metal complex linker and cocatalyst for enhanced photocatalytic activity via g-C3N4 hybrid

[{Cu(phen)}2(μ-OH)2(Ag3Br5)]n (1)/g-C3N4 composite was prepared by in situ growth under solvothermal conditions. The composite exhibited much higher photocatalytic efficiency than the parents 1 and g-C3N4 in the degradation of crystal violet.

Conjugation site characterization of antibody–drug conjugates using electron-transfer/higher-energy collision dissociation (EThcD)

Antibody–drug conjugates (ADCs) are formed by binding of cytotoxic drugs to monoclonal antibodies (mAbs) through chemical linkers. A comprehensive evaluation of the critical quality attributes (CQAs) of ADCs is vital for drug development but remains challenging owing to ADC structural heterogeneity than mAbs. Drug conjugation sites can considerably affect ADC properties, such as stability and pharmacokinetics, however, few studies have focused on method development in this area owing to technical challenges. Hybrid electron-transfer/higher-energy collision dissociation (EThcD) produces more fragment ions than conventional higher-energy collision dissociation (HCD) fragmentation, which aids in identifying and localizing post-translational modifications. Herein, we systematically employ EThcD to assess the fragmentation mode impact on conjugation site characterization for randomly conjugated and site-specific ADCs. EThcD generates more fragment ions in tandem mass spectrometry (MS/MS) spectra compared with HCD. Additional ions aid in pinpointing the correct conjugation sites that bear complex linker payload structures. Our study may contribute to the quality control of various preclinical and clinical ADCs.

Combined study of docking and molecular dynamics against DNV-3 SN1 protein by bixinoids

Dengue (DENV), Zica virus (ZIKV), and Chikungunya fever (CHIK) are tropical diseases that have caused a lot of problems in general worldwide. Transmitted by mosquitoes of the species Aedes aegypti and albopictus, they have not been completely eradicated in the country, and their proliferation has only increased in the Northeast region. Within the structure of the virus, it is possible to verify the presence of glycoprotein SN1, which is responsible for its replication. If this macromolecule is inhibited using a specific or complex linker, it can interrupt its replication activity. An alternative to this problem has been using structures derived from natural products that have pharmacological properties. A dynamic and molecular docking combined study used computational simulation in the four isomeric forms of bixin against the SN1 protein. The Z,E-bixin and E,E-bixin isomers, both with affinity energy −6.7 and −6.5 Kcal/mol, presented the best results. Thus, bixin and its isomers, found in annatto seeds, maybe an initial proposal in the search for prototype compounds to study to fight this lethal virus in the future. Communicated by Ramaswamy H. Sarma

Estimation of Effective Concentrations Enforced by Complex Linker Architectures from Conformational Ensembles.

Proteins and protein assemblies often tether interaction partners to strengthen interactions, to regulate activity through auto-inhibition or -activation, or to boost enzyme catalysis. Tethered reactions are regulated by the architecture of the tether, which defines an effective concentration of the interactor. Effective concentrations can be estimated theoretically for simple linkers via polymer models, but there is currently no general method for estimating effective concentrations for complex linker architectures consisting of both flexible and folded domains. We describe how effective concentrations can be estimated computationally for any protein linker architecture by defining a realistic conformational ensemble. We benchmark against prediction from a worm-like chain and values measured by competition experiments and find minor differences likely due to excluded volume effects. Systematic variation of the properties of flexible and folded segments shows that the effective concentration is mainly determined by the combination of the total length of flexible segments and the distance between the termini of the folded domains. We show that a folded domain in a disordered linker can increase the effective concentration beyond what can be achieved by a fully disordered linker by focusing the end-to-end distance at the appropriate spacing. This suggests that complex linker architecture may have advantages over simple flexible linkers and emphasizes that annotation as a linker should depend on the molecular context.

Image-based high-throughput mapping of TGF-\u03b2-induced phosphocomplexes at a single-cell level

Protein interactions and posttranslational modifications orchestrate cellular responses to e.g. cytokines and drugs, but it has been difficult to monitor these dynamic events in high-throughput. Here, we describe a semi-automated system for large-scale in situ proximity ligation assays (isPLA), combining isPLA in microtiter wells with automated microscopy and computer-based image analysis. Phosphorylations and interactions are digitally recorded along with subcellular morphological features. We investigated TGF-β-responsive Smad2 linker phosphorylations and complex formations over time and across millions of individual cells, and we relate these events to cell cycle progression and local cell crowding via measurements of DNA content and nuclear size of individual cells, and of their relative positions. We illustrate the suitability of this protocol to screen for drug effects using phosphatase inhibitors. Our approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput.

Boron doped diamond thin films for the electrochemical detection of SARS-CoV-2 S1 protein

Amidst a global pandemic, a precise and widely accessible rapid detection method is needed for accurate diagnosis and contact tracing. The lack of this technology was exposed through the outbreak of SARS-CoV-2 beginning in 2019. This study sets the foundation for the development of a boron doped diamond (BDD)-based impedimetric sensor. While specifically developed for use in the detection of SARS-CoV-2, this technology uses principles that could be adapted to detect other viruses in the future. Boron doped polycrystalline diamond electrodes were functionalized with a biotin-streptavidin linker complex and biotinylated anti-SARS-CoV-2 S1 antibodies. Electrodes were then incubated with the S1 subunit of the SARS-CoV-2 spike surface protein, and an electrical response was recorded using the changes to the electrode's charge transfer resistance (Rct), measured through electrochemical impedance spectroscopy (EIS). Detectable changes in the Rct were observed after 5-min incubation periods with S1 subunit concentrations as low as 1 fg/mL. Incubation with Influenza-B Hemagglutinin protein resulted in minimal change to the Rct, indicating specificity of the BDD electrode for the S1 subunit of SARS-CoV-2. Detection of the S1 subunit in a complex (cell culture) medium was also demonstrated by modifying the EIS protocol to minimize the effects of sample matrix binding. BDD films of varying surface morphologies were investigated, and material characterization was used to give insight into the microstructure-performance relationship of the BDD sensing surface.

Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity.

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.

Photoactive Zr and Ti Metal‐Organic‐Frameworks for Solid‐State Solar Cells

Solid-state photovoltaic cells based on robust metal-organic frameworks (MOFs), MIL-125(Ti), MIL-125(Ti)-NH2 , UiO-67, Ru(bpy)2 -UiO-67, (bpy 2,2'-bipyridine) as active components and spiro-MeOTAD (MeOTAD 2,2',7,7'-tetrakis[N,N-di(p-methoxyphenyl)amino]-9,9'-spirobifluorene) as hole transporting layer have been prepared., The photovoltaic response of this material increases in the presence of bathochromic -NH2 groups on the linker or Ru (II) polypyridyl complexes light harvester. These results show that the strategies typically employed in photocatalysis to enhance the photocatalytic activity of MOFs can also be applied in the field of photovoltaic devices.

Z-Ring-Associated Proteins Regulate Clustering of the Replication Terminus-Binding Protein ZapT in Caulobacter crescentus.

Regulated organization of the chromosome is essential for faithful propagation of genetic information. In the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is spatially arranged in close proximity to the cytokinetic Z-ring during the cell cycle. Although the Z-ring-associated proteins ZapA and ZauP interact with the terminus recognition protein ZapT, the molecular functions of the complex that physically links the terminus and the Z-ring remain obscure. In this study, we found that the physical linkage helps to organize the terminus DNA into a clustered structure. Neither ZapA nor ZauP was required for ZapT binding to the terminus DNA, but clustering of the ZapT-DNA complexes over the Z-ring was severely compromised in cells lacking ZapA or ZauP. Biochemical characterization revealed that ZapT, ZauP, and ZapA interacted directly to form a highly ordered ternary complex. Moreover, multiple ZapT molecules were sequestered by each ZauP oligomer. Investigation of the functional structure of ZapT revealed that the C terminus of ZapT specifically interacted with ZauP and was essential for timely positioning of the Z-ring in vivo Based on these findings, we propose that ZauP-dependent oligomerization of ZapT-DNA complexes plays a distinct role in organizing the replication terminus and the Z-ring. The C termini of ZapT homologs share similar chemical properties, implying a common mechanism for the physical linkage between the terminus and the Z-ring in bacteria.IMPORTANCE Rapidly growing bacteria experience dynamic changes in chromosome architecture during chromosome replication and segregation, reflecting the importance of mechanisms that organize the chromosome globally and locally within a cell to maintain faithful transmission of genetic material across generations. During cell division in the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is physically linked to the cytokinetic Z-ring at midcell. However, the functions of this physical linkage are not fully understood. We adopted biochemical and cell-biological techniques to characterize the linkage, including the terminus-binding protein ZapT and the Z-ring-associated protein ZauP. We obtained evidence that the Z-ring organizes the chromosome terminus into a compact structure at midcell via specific interaction between ZapT and ZauP oligomers. Because these proteins are conserved in diverse Gram-negative bacteria, our findings highlight a novel and conserved role for the linker complex in regulated organization of the chromosome terminus.

Mechanistic Insights into Regulation of the ALC1 Remodeler by the Nucleosome Acidic Patch

Upon DNA damage, the ALC1/CHD1L nucleosome remodeling enzyme (remodeler) is activated by binding to poly(ADP-ribose). How activated ALC1 recognizes the nucleosome, as well as how this recognition is coupled to remodeling, is unknown. Here, we show that remodeling by ALC1 requires a wild-type acidic patch on the entry side of the nucleosome. The cryo-electron microscopy structure of a nucleosome-ALC1 linker complex reveals a regulatory linker segment that binds to the acidic patch. Mutations within this interface alter the dynamics of ALC1 recruitment to DNA damage and impede the ATPase and remodeling activities of ALC1. Full activation requires acidic patch-linker segment interactions that tether the remodeler to the nucleosome and couple ATP hydrolysis to nucleosome mobilization. Upon DNA damage, such a requirement may be used to modulate ALC1 activity via changes in the nucleosome acidic patches.

Crowning the Kinetochore: The Fibrous Corona in Chromosome Segregation.

The kinetochore is at the heart of chromosome segregation in mitosis and meiosis. Rather than a static linker complex for chromatin and spindle microtubules, it is highly dynamic in composition, size, and shape. While known for decades that it can expand and grow a fibrous meshwork known as the corona, it was until recently unclear what constitutes this 'crown' and what its relevance is for kinetochore function. Here, we highlight recent discoveries in fibrous corona biology, and place them in the context of the processes that orchestrate high-fidelity chromosome segregation.

Peptide AEDL alters chromatin conformation via histone binding

Eukaryotic DNA is tightly packed into chromatin, a DNA–protein structure that exists as transcriptionally permissive euchromatin or repressive heterochromatin. Post-translational modification of histones plays a key role in regulating chromatin dynamics. Short peptides derived from various sources are known to function as epigenetic modulators; however, their mechanisms of action are poorly understood. We addressed this issued by investigating the effect of peptide AEDL on chromatin structure in tobacco (Nicotiana tabacum L.), a commercially important plant species. The chromatin of tobacco interphase cells is characterized by the presence of zones of transcriptionally active domains and particular domains of condensed chromatin of cells that partially coincide with heterochromatin zones. Chromatin decondensation and the formation of euchromatin, accompanied by the activation of genes expression activity, are a determining factor in responses to stressful effects. Our results show that plants grown in the presence of 10⁻⁷ M peptide AEDL transformed condensed chromatin domains from 45% in control cells to 25%. Histone modifications, which constitute the so-called histone code, play a decisive role in the control of chromatin structure. Fluorescence quenching experiments using fluorescein isothiocyanate-labeled histones revealed that the linker histone H1 and complexes of core H3 and H1 histones with DNA bound to peptide AEDL in a 1: 1 molar ratio. The peptide was found to bind to the N-terminal lysine residue of H1 and the lysine residue at position 36 of the H3 C terminus. These interactions of histones H1 and H3 with AEDL peptide loosened the tightly packed chromatin structure, getting transcriptionally active euchromatin. Our findings provide novel insight into the mechanism of gene regulation by short peptides and have implications for breeding more resistant or productive varieties of tobacco and other crops.

Control of Porphyrin Dye Aggregation Using Bis(4-pyridyl)Alkanes in Dye Sensitized Solar Cells

The aggregation of sensitizer molecules on the surface of photoanode is a serious issue that can affect the photovoltaic performance of dye-sensitized solar cells. Prevention of dye agglomeration, therefore, is critical. Traditional methods of aggregation control are either synthetically challenging or technologically difficult and expensive. In this article, the use of bis(4-pyridyl)alkanes to control porphyrin dye aggregation is presented. Three bis(4-pyridyl)alkanes – bis(4-pyridyl)butane L4, bis(4-pyridyl)octane L8 and bis(4-pyridyl)decane L10 were synthesized. These bis(4-pyridyl)alkane ligands are axially attached to the metallic center of synthesized porphyrin dye P. The complexes was obtained by mixing the solutions of dye P and each ligand (L) in 2:1 ratio 1 h before the soaking step. As a result three cells were prepared: P-L4, P-L8 and P-L10. The performance of these cells were compared with a reference cell which was prepared from porphyrin dye P only. IPCE analysis demonstrated the highest dye load in P-L4 cell which was ascribed to lowered dye aggregation. Photovoltaic analysis showed improved short circuit current density due to suppressed dye aggregation caused by the complexation of the porphyrin dye P with the linker L4. As a result the overall cell efficiency increased to 42% demonstrating the successful utilization of the (4-pyridyl)alkane linker complexes with porphyrin dye.

Radio-pharmaceuticals for cancer treatment: are they ready for prime time yet?

Radio-pharmaceuticals for cancer treatment: are they ready for prime time yet?

Salicylamide derivatives for iron and aluminium sequestration. From synthesis to complexation studies

This paper presents an easy, fast and economic synthesis of chelating agents for medical, environmental and analytical applications, and the evaluation of the stability of their complexes with Fe3+ and Al3+. Complex formation equilibria with Cu2+ and Zn2+ metal ions were also studied to evaluate if the chelating agents can perturb the homeostatic equilibria of these essential metal ions. Effective chelating agents for metal ions, in addition to their well-known medical uses, find an increasing number of applications in environmental remediation, agricultural applications (supplying essential elements in an easily available form), and in analytical chemistry as colorimetric reagents. Besides the stability of the complexes, the lack of toxicity and the low cost are the basic requisites of metal chelating agents. With these aims in mind, we utilized ethyl salicylate, a cheap molecule without toxic effects, and adopted a simple synthetic strategy to join two salicylate units through linear diamines of variable length. Actually, the mutual position of the metal binding oxygen groups, as well as the linker length, affected protonation and complex formation equilibria. A thorough study of the ligands is presented. In particular, the complex formation equilibria of the three ligands toward Fe3+, Al3+, Zn2+ and Cu2+ ions were investigated by combined potentiometric and spectrophotometric techniques. The results are encouraging: all the three ligands form stable complexes with all the investigated metal ions, involving the oxygen donor atoms from the 2-hydroxybenzamido unit, and nitrogen atoms in copper and zinc coordination.

A 3-D Wells–Dawson phosphotungstate hybrid material modified by Na+ and a bi-copper organic complex linker, and its photo-/electro-catalytic properties

An inorganic–organic hybrid material based on Dawson polyoxometalates, [Na2{CuI2(bim)2}2(P2W18O62)]·2H2O (bim = biimidazole) (1), has been hydrothermally synthesized and fully characterized. In 1, each {P2W18} cluster as a hexa-node ligand connects to six adjacent Dawson clusters via six Na1 ions to generate a pure inorganic 2-D layer, further joined by bimetal–organic segments [Cu2(bim)2]2+ to form a 3-D inorganic–organic hybrid framework with channels. The structure can be simplified to 3,8-connected 3-D networks with (43)(418·624·83) topology, which shows bifunctional electrocatalytic performance for oxidation of ascorbic acid (AA) and reduction of nitrite (NO2−), and high-efficient degradation ability for three typical dyes under UV light irradiation.

Cytoskeleton and nucleoskeleton involvement in processes of cytomixis in plants.

Cytomixis is a form of cell-to-cell nuclear migration that involves the interaction of dynamic cytoskeletal components with the nucleus through signalling systems and linker complexes. In cytomixis two known mechanisms can be involved: actomyosin and/or microtubules and their associated motors. Perinuclear actin anchors and determines the direction of nuclear movement. In microsporogenesis cytomixis is probably initiated by a cascade of signals that trigger prophase reorganization of nucleus and cytoskeleton, and is a result of cytoskeletal protein activation, as well as a weakening of mechanisms responsible for anchoring the nucleus. The interactions between nuclei and the cytoskeleton are mediated by linker complexes that play a major role in nuclear positioning and shape, chromatin-nuclear envelope interactions, nucleoskeleton organization, gene expression and genome organization. Other contributing factors include changes in the protein composition and post-translational modifications that alter protein conformation. Cytomixis appears also to have relevance to higher order structuring, influencing tissue and organ architecture, causing collective forms of cell interactions and information exchange within a single continuum. In this review we summarize our current understanding of the cytoskeleton dynamic function in cytomictic nuclear migration.

Cellular mechanisms of nuclear migration

Subcellular mobility, positioning, and directional movement of the nucleus in a certain site of the cell or cenocyte and, less frequently, intercellular translocation of the nucleus accompany the cell and tissue differentiation, change of their functions, and the organism growth and development and its response to stress, plant–microbial interactions, symbiosis, and many other processes in plants and animals. The nucleus movement is performed and directed through the interaction between dynamic cytoskeleton components and nucleus by means of signal-binding proteins, including motor and linker. The cell responds to the external signal by mobilization and polar reconstruction of the cytoskeleton components, as a result of which the nucleus displacement by means of actomyosin or microtubule mechanisms in cooperation with dynein and kinesin occurs. In plants, the actomyosin mechanism is involved in the nucleus migration; it allows the nucleus to move rapidly and over significant distances in response to environmental stimuli. An important role in the nucleus translocation belongs to the linker complexes of the proteins that are inserted in the nuclear envelope, that connect and transmit signals from the plasmalemma to the cytoplasm and nucleoplasm, and that provide the skeletal basis for many subcellular compartments. Changes in the protein composition, conformational modifications of the proteins, and displacement of linkers from the nuclear envelope result in the nucleus detachment from the cytoskeleton, and change in the form, mechanical rigidity, and positioning of the nucleus.

Detection of Rota Virus with the Help of Nanomaterial Based Field Effect Transistor (BIO-FET)

Biosensors are analytical devices composed of a recognition element of biological origin and a physiochemical transducer which converts the reaction into readable signals. The biosensors are providing their application in medical diagnosis and clinical analysis from last fifty years. In this work we have synthesized reduced graphene oxide, with this nano-film of RGO a FET- biosensor was developed and tested for the rapid detection of Rota-virus. It is a sensitive, specific and label free detection protocol. In the present methodology graphene; which is a single monomer unit of graphite was used and fabricated between the 3 μm apart micro-fabricated Au-electrode over hot plate at 70°C on a crystal surface by drop casting method. This thin film of graphene on FET crystal provide a better platform for attachment of antibodies through pyrene-NHS (Linker) complex. Pyrene- NHS is identified more suitable for antibody immobilization on the graphene film to be used in nano-immunosensor for the detection of specific antigens. The Rota-virus interation to its corresponding antibody were studied with respect to changes in the conductance in the graphene channel. This chemi-resistive FET transducer based 1D nanostructure have attracted much attention because of its rapid, sensitive, and cost effective point of care device for quantitative detection of Rota-virus so that gastroenteritis in infants can be detected early. The graphene nanofilm has been characterized by UV-Vis Spectroscopy, FT-IR, EDS, and SEM.