Reversible Complex Research Articles

TCR-ligand dissociation rate is a robust and stable biomarker of CD8+ T cell potency.

Despite influencing many aspects of T cell biology, the kinetics of T cell receptor (TCR) binding to peptide-major histocompatibility molecules (pMHC) remain infrequently determined in patient monitoring or for adoptive T cell therapy. Using specifically designed reversible fluorescent pMHC multimeric complexes, we performed a comprehensive study of TCR-pMHC off-rates combined with various functional assays on large libraries of self/tumor- and virus-specific CD8+ T cell clones from melanoma patients and healthy donors. We demonstrate that monomeric TCR-pMHC dissociation rates accurately predict the extent of cytotoxicity, cytokine production, polyfunctionality, cell proliferation, activating/inhibitory receptor expression, and in vivo antitumor potency of naturally occurring antigen-specific CD8+ T cells. Our data also confirm the superior binding avidities of virus-specific T cells as compared with self/tumor-specific T cell clonotypes (n > 300). Importantly, the TCR-pMHC off-rate is a more stable and robust biomarker of CD8+ T cell potency than the frequently used functional assays/metrics that depend on the T cell's activation state, and therefore show major intra- and interexperimental variability. Taken together, our data show that the monomeric TCR-pMHC off-rate is highly useful for the ex vivo high-throughput functional assessment of antigen-specific CD8+ T cell responses and a strong candidate as a biomarker of T cell therapeutic efficacy.

Fundamentally Addressing Bromine Storage through Reversible Solid-State Confinement in Porous Carbon Electrodes: Design of a High-Performance Dual-Redox Electrochemical Capacitor

Research in electric double-layer capacitors (EDLCs) and rechargeable batteries is converging to target systems that have battery-level energy density and capacitor-level cycling stability and power density. This research direction has been facilitated by the use of redox-active electrolytes that add faradaic charge storage to increase energy density of the EDLCs. Aqueous redox-enhanced electrochemical capacitors (redox ECs) have, however, performed poorly due to cross-diffusion of soluble redox couples, reduced cycle life, and low operating voltages. In this manuscript, we propose that these challenges can be simultaneously met by mechanistically designing a liquid-to-solid phase transition of oxidized catholyte (or reduced anolyte) with confinement in the pores of electrodes. Here we demonstrate the realization of this approach with the use of bromide catholyte and tetrabutylammonium cation that induces reversible solid-state complexation of Br2/Br3-. This mechanism solves the inherent cross-diffusion issue of redox ECs and has the added benefit of greatly stabilizing the reactive bromine generated during charging. Based on this new mechanistic insight on the utilization of solid-state bromine storage in redox ECs, we developed a dual-redox EC consisting of a bromide catholyte and an ethyl viologen anolyte with the addition of tetrabutylammonium bromide. In comparison to aqueous and organic electric double-layer capacitors, this system enhances energy by factors of ca. 11 and 3.5, respectively, with a specific energy of ∼64 W·h/kg at 1 A/g, a maximum power density >3 kW/kg, and cycling stability over 7000 cycles.

Molecular determinants for cyclo-oligosaccharide-based nanoparticle-mediated effective siRNA transfection.

To study the structural requirements that a cyclooligosaccharide-based nanoparticle must fulfill to be an efficient siRNA transfection vector. siRNA protection from degradation by RNAses, transfection efficiency and the thermodynamic parameters of the nanoparticle/siRNA interactions were studied on pairs of amphiphilic molecules using biochemical techniques and molecular dynamics. The lower the siRNA solvent accessible surface area in the presence of the nanoparticle, higher the protection from RNAse-mediated degradation in the corresponding nanocomplex; a moderate nanoparticle/siRNA binding energy value further facilitates reversible complexation and binding to the target cellular mRNA. The use, in advance, of these parameters will provide a useful indication of the potential of a molecular nanoparticle as siRNA transfecting vector.

Selective and Reversible Fluoride Complexation from Water by a Cyclic Tri(phosphonio)methanide Dication.

Tri(phosphonio)methanide dication 32+ , prepared from a trifluoromethylsulfanylphosphonium dication (12+ ) via an intramolecular electrophilic aromatic substitution reaction, is an unexpected P-based, water-resistant Lewis acid that is capable to selectively and reversibly bind fluoride ions from organic/aqueous biphasic solution. The formed complex is an unusual fluorophosphorane ([3-F]OTf). The multiple donor-acceptor interactions of 32+ that are crucial for the fluoride fixation have been elucidated by quantum chemical calculation. Compound [3-F]OTf can also be used as a convenient anhydrous fluoride ion source and was probed as a suitable catalyst of the silylotrifluoromethylation of various aldehydes.

Selective and Reversible Fluoride Complexation from Water by a Cyclic Tri(phosphonio)methanide Dication

AbstractTri(phosphonio)methanide dication 32+, prepared from a trifluoromethylsulfanylphosphonium dication (12+) via an intramolecular electrophilic aromatic substitution reaction, is an unexpected P‐based, water‐resistant Lewis acid that is capable to selectively and reversibly bind fluoride ions from organic/aqueous biphasic solution. The formed complex is an unusual fluorophosphorane ([3‐F]OTf). The multiple donor–acceptor interactions of 32+ that are crucial for the fluoride fixation have been elucidated by quantum chemical calculation. Compound [3‐F]OTf can also be used as a convenient anhydrous fluoride ion source and was probed as a suitable catalyst of the silylotrifluoromethylation of various aldehydes.

Structural investigation of the HIV-1 reverse transcriptase initiation complex by HDX, SAXS, CryoEM and X-ray crystallography

Structural investigation of the HIV-1 reverse transcriptase initiation complex by HDX, SAXS, CryoEM and X-ray crystallography

Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance.

HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATP (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.

Imaging HIV-1 Genomic DNA from Entry through Productive Infection.

In order to track the fate of HIV-1 particles from early entry events through productive infection, we developed a method to visualize HIV-1 DNA reverse transcription complexes by the incorporation and fluorescent labeling of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) into nascent viral DNA during cellular entry. Monocyte-derived macrophages were chosen as natural targets of HIV-1, as they do not divide and therefore do not incorporate EdU into chromosomal DNA, which would obscure the detection of intranuclear HIV-1 genomes. Using this approach, we observed distinct EdU puncta in the cytoplasm of infected cells within 12 h postinfection and subsequent accumulation of puncta in the nucleus, which remained stable through 5 days. The depletion of the restriction factor SAMHD1 resulted in a markedly increased number of EdU puncta, allowing efficient quantification of HIV-1 reverse transcription events. Analysis of HIV-1 isolates bearing defined mutations in the capsid protein revealed differences in their cytoplasmic and nuclear accumulation, and data from quantitative PCR analysis closely recapitulated the EdU results. RNA fluorescence in situ hybridization identified actively transcribing, EdU-labeled HIV-1 genomes in productively infected cells, and immunofluorescence analysis confirmed that CDK9, phosphorylated at serine 175, was recruited to RNA-positive HIV-1 DNA, providing a means to directly observe transcriptionally active HIV-1 genomes in productively infected cells. Overall, this system allows stable labeling and monitoring of HIV genomic DNA within infected cells during cytoplasmic transit, nuclear import, and mRNA synthesis.IMPORTANCE The fates of HIV-1 reverse transcription products within infected cells are not well understood. Although previous imaging approaches identified HIV-1 intermediates during early stages of infection, few have connected these events with the later stages that ultimately lead to proviral transcription and the production of progeny virus. Here we developed a technique to label HIV-1 genomes using modified nucleosides, allowing subsequent imaging of cytoplasmic and nuclear HIV-1 DNA in infected monocyte-derived macrophages. We used this technique to track the efficiency of nuclear entry as well as the fates of HIV-1 genomes in productively and nonproductively infected macrophages. We visualized transcriptionally active HIV-1 DNA, revealing that transcription occurs in a subset of HIV-1 genomes in productively infected cells. Collectively, this approach provides new insights into the nature of transcribing HIV-1 genomes and allows us to track the entire course of infection in macrophages, a key target of HIV-1 in infected individuals.

Overlay-independent comparisons of X-ray structures reveal small, systematic conformational changes in liganded acetylcholinesterase

Background and Purpose: Comparisons of macromolecular 3D structures by minimizing corresponding atom RMSDs can by virtue of averaging, hide small unexpected deviations caused by ligand binding or site-directed mutagenesis. We developed an overlay-independent comparison approach and used it to compare selected, PDB structures of acetylcholinesterase (AChE; EC 3.1.1.7) in the apo state, in complexes with reversible ligands, in covalent conjugates or upon AChE mutagenesis. Methods: The active serine alpha carbon of the AChE monomer was used as a frame of reference for each of the structures, and distances calculated to remaining alpha carbons of the monomer backbone. Differences in distances at corresponding backbone positions from pairwise comparisons with apo-AChE structures were taken as a measure of changes in tertiary structures of liganded or mutant AChE monomers.Results: This analysis revealed small, yet significant backbone shifts in monomeric tertiary structures of AChEs. While reversible complexes with neurotoxin Fas2 showed global expansion of AChE monomer backbones, covalent conjugates at the active serine and high affinity reversible inhibitor hupA complexes revealed backbone compression towards the active serine. In some of AChE homodimers 0.6 - 1Å shifts of monomer backbone affected crystallographic quaternary structures by causing significant, up to seven degree tilts in monomers, relative to angles measured in the apo-AChE.Conclusions: Small but systematic differences detected in tertiary structures of liganded or mutagenized AChEs appear consistent with larger magnitude alterations of several AChE homodimer quaternary structures. Those observations vaildate our overlay-independent approach for detecting small, yet significant, tertiary backbone shifts in macromolecular 3D structures.

Design of Organic Macrocycle-Modified Iron Oxide Nanoparticles for Drug Delivery.

Paul Ehrlich's vision of a "magic bullet" cure for disease inspires the modern design of nanocarriers whose purpose is to deliver drug cargo to specific sites in the body while circumventing endogenous immunological clearance mechanisms. Iron oxide nanoparticles (IONPs) have emerged as particularly promising nanocarriers because of their biodegradability, ability to be guided magnetically to sites of pathology, mediation of hyperthermic therapy, and imaging capabilities. In this review, we focus on the design and drug-delivery aspects of IONPs coated with organic macrocycles (crown ethers, cyclodextrins, calix[n]arenes, cucurbit[n]urils, or pillar[n]arenes), which, by means of reversible complexation, allow for the convenient loading and release of drug molecules. Macrocycles can be attached to IONPs indirectly or directly. Indirect attachment requires the use of small organic linking molecules or conjugation to shell materials. Direct attachment requires neither. We discuss in detail drug release from the macrocycles, highlighting mechanisms that depend on external stimuli such as changes in pH, the competitive binding of ions or small molecules, or the application of ultrasound or electromagnetic radiation.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3' ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Molecular Mechanism of Inhibition of DNA Methylation by Zebularine

In this work, we have analyzed the molecular mechanism of inhibition of a C5-DNA methyltransferase by zebularine using classical and QM/MM simulations. We found that the reaction proceeds with the addition of an unprotonated cysteine to the C6 position of the ring followed by methyl transfer to the C5 position. However, while the first step is reversible and presents a moderate free-energy barrier, the second step presents a large free-energy barrier, preventing the formation of the methylated complex. This mechanistic proposal agrees with recent experimental observations that point to the formation of a reversible covalent complex between DNA containing zebularine and methyltransferases. The absence of the exocyclic amino group in zebularine, as compared to the natural substrate cytosine, makes it more difficult to reach an optimal orientation of the substrate both for cysteine addition and methyl transfer and decreases the nucleophilicity of the carbon atom to be methylated, making this step unaffordabl...

A General Framework for the Boundary Structure in Multi-Component Sedimentation Velocity with Reversible Interactions

The coupled transport behavior of macromolecular mixtures with rapidly reversible complex formation is of great interest in the study of protein interactions by many different methods. Complicated transport patterns arise even for simple bimolecular reactions, when all species exhibit different migration velocities, yet are coupled by mass action law. Although partial differential equations are available to describe the spatio-temporal evolution of such systems given particular initial conditions, a general overview of their behavior, for example, as a function of concentration, velocities, and binding constants, has not yet been reported. For two-component systems, based on the limit of non-diffusing species migrating under constant force, we have previously derived simple algebraic relationships describing the number, amplitude, and velocity of sedimentation boundaries, which are consistent with the principle that the time-average velocity of all co-sedimenting component fractions in each boundary must match. In combination, these can be considered ‘effective particles’, and their properties can be related to parameters extracted from experimental sedimentation profiles of mixtures of two interacting protein components. To support the study of three-component (and higher) interacting protein mixtures, here, we generalize this effective particle theory to N-component systems. These can have N-2 reaction boundaries of successively lower velocity and number of components. We report algebraic relationships for velocities and compositions. Considering the space of total component concentrations, we show it is subdivided into regions resulting in different boundary patterns, separated by discontinuous transitions along characteristic surfaces. These meet in a single point - a unique combination of component concentrations where the entire system can migrate as if it was a single species. The extension of the effective particle theory provides physical insights into the coupled co-migration processes, and we believe this will be useful to interpret experimentally robust parameters derived from measured sedimentation coefficient distributions of multi-component mixtures with rapidly reversible interactions.

Cooperative Reinforcement of Ionic Liquid and Reactive Solvent on Enzymatic Synthesis of Caffeic Acid Phenethyl Ester as an In Vitro Inhibitor of Plant Pathogenic Bacteria.

It is widely believed that lipases in ionic liquids (ILs) possess higher enzyme activity, stability and selectivity; however, reaction equilibrium is always limited by product inhibition, and the product is difficult to separate from non-volatile ILs using distillation. To solve this problem, using trialkylphosphine oxide (TOPO) as a complexing agent, a novel biphase of reactive solvent and IL was firstly reported for caffeic acid phenethyl ester (CAPE) production from methyl caffeate (MC) and 2-phenylethanol (PE) catalyzed by lipase via transesterification. The effects of the reaction parameters and their action mechanism were investigated, and the inhibition of CAPE against bacterial wilt pathogen Ralstonia solanacearum was firstly measured. The MC conversion of 98.83% ± 0.76% and CAPE yield of 96.29% ± 0.07% were obtained by response surface methodology in the 25 g/L TOPO-cyclohexane/[Bmim][Tf2N] (1:1, v/v); the complex stoichiometry calculation and FTIR spectrum confirmed that the reversible hydrogen-bond complexation between TOPO and caffeates significantly enhances the cooperative effect of two phases on the lipase-catalyzed reaction. The temperature was reduced by 14 °C; the MC concentration increased by 3.33-fold; the ratio of catalyst to donor decreased by 4.5-fold; and Km decreased 1.08-fold. The EC50 of CAPE against R. solanacearum was 0.17–0.75 mg/mL, suggesting that CAPE is a potential in vitro inhibitor of plant pathogenic bacteria.

Сitric acid influence on electroreduction processes in low concentrated Cu(NO3)2 solutions

Physico-chemical regularities citric acid influence on processes of copper and nitrate-ions reduction in low concentrated nitrate solutions were established. An influence of pH value on inhibitor action of low concentrations of the additive according to cathode reduction reaction of nitrate-ions, on kinetics of copper electrodeposition, on structure of the precipitates obtained was shown. Polarization measurements show us changes of copper electrodeposition at introduction of citric acid in Cu(NO3)2 solution. By the methods of traced elements and extraction into aqueous phase the reversible inclusion complexes into electrolytic copper, was established that proves participation of complexes in cathode process.

Hybrid pectin-Fe3+/polyacrylamide double network hydrogels with excellent strength, high stiffness, superior toughness and notch-insensitivity.

The lack of sufficient mechanical properties restricts the application of polysaccharide-based hydrogels in the field of biomedicine, especially load-bearing tissue repair. Nowadays, double network (DN) hydrogels have aroused great interest through special cooperation between two contrasting networks. Inspired by this idea, here, we devised a new strategy to prepare a pectin-Fe3+/polyacrylamide hybrid DN hydrogel using a simple two-step method. The introduction of Fe3+ ions into a pectin network to produce strong reversible ionic complexation, results in excellent toughness. Under optimal conditions, our hybrid DN hydrogels possessed tensile strength as high as 0.9 MPa, corresponding to a high strain of 1300%. Besides, our hybrid DN hydrogels also exhibited superb stiffness (elastic modulus ∼ 1.46 MPa), toughness (fracture energy ∼ 3785 J m-2), and water absorption capacity (85%). Loading-unloading tests showed that the internal fracture process of the hydrogels was continuous. Owing to the reversible structure of Fe3+-pectin complexation, the hybrid DN hydrogels also showed good fatigue resistance, notch-insensitivity and recoverability. This type of polysaccharide-based hydrogel has potential to broaden the application in the load-bearing tissue repair field.

Effect of polymer topology on non-covalent polymer–protein complexation: miktoarm versus linear mPEG-poly(glutamic acid) copolymers

We report the design of mPEG-(poly(glutamic acid)) with different macromolecular topology – linear and miktoarm – for reversible non-covalent protein complexation.

Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry.

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA atreplication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to thetime of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.

Bismuth Complexes in Phenylazomethine Dendrimers: Controllable Luminescence and Emission in the Solid State

Dendritic phosphors were obtained by the stepwise integration of BiCl3 in phenylazomethine dendrimers. The bismuth-coordinated phenylazomethines displayed photoluminescence at 500-800 nm, and the intensity could be tuned by changing the stoichiometry of BiCl3 and the dendrimer. This phosphor did not show serious luminescence quenching even though the local concentration of BiCl3 in the dendrimer was as high as 20 M, and luminescence was also observed in the solid state. The absorption and emission properties could be reversibly switched by addition of a Lewis base or under electrochemical redox control, which induced the reversible complexation of BiCl3 in the dendrimer.

Celecoxib non-competitively inhibits acetylcholinesterase

Isocitrate lyase from the mycelium of Phycomyces blakesleeanus was inactivated with thiol-reactive reagents, 5,5′-dithiobis-(2-nitrobenzoic)acid, p-hydroxymercuribenzoic acid, N-ethylmaleimide or iodoacetate, at pH 6.8 and 25°C. In all cases the inactivation is characterized by a biphasic kinetic profile. The rapid initial phase of inactivation does not increase linearly with increasing reagent concentration, but exhibits an apparent saturation effect, suggesting the formation of a reversible complex between the enzyme and the reagent prior to the inactivation step. Re-activation of the enzyme was observed under thiol excess treatment. The pH dependence of the initial phase of inactivation suggests that a group on the enzyme with pKa = 6.8 is being modified. The effect of ligands was tested on the inactivation reaction. Mg2+-Ds-isocitrate and Ds-isocitrate provided total protection, whereas Mg2+ ions, succinate and oxalate provided only partial protection of the enzyme against inactivation. On the basis of these results, we would suggest that the thiol-reactive reagents modify at least one thiol group crucial for the enzymatic activity and probably located in the interface between succinate and glyoxylate subsite.