Tetrameric Molecule Research Articles

Crystal structure of the flavin-dependent thymidylate synthase Thy1 from Thermus thermophilus with an extra C-terminal domain

The thymidylate synthases ThyA and Thy1 are enzymes that catalyse the formation of thymidine monophosphate from 2'-deoxyuridine monophosphate. Thy1 (or ThyX) requires flavin for catalytic reactions, while ThyA does not. In the present study, the crystal structure of the flavin-dependent thymidylate synthase Thy1 from Thermus thermophilus HB8 (TtThy1, TTHA1096) was determined in complex with FAD and phosphate at 2.5 Å resolution. TtThy1 is a tetrameric molecule like other Thy1 proteins, to which four FAD molecules are bound. In the crystal of TtThy1, two phosphate ions were bound to each dUMP-binding site. The characteristic feature of TtThy1 is the existence of an extra C-terminal domain (CTD) consisting of three α-helices and a β-strand. The function of the CTD is unknown and database analysis showed that this CTD is only shared by part of the Deinococcus-Thermus phylum.

Formation of Polymeric Housene Molecules of Group 15 Elements (N, P, As, and Sb): A DFT Study.

Recently, the formation of the dimeric stibahousene molecule, bis(stibahousene), has been reported. In line with the report, the formation of dimeric housene molecules with N, P, and As is examined in light of density functional theory. Moreover, the extension of the study from dimeric to tetrameric and hexameric molecules (N, P, As, and Sb) is also performed. The study supports the formation of such polymeric housene analogues.

Molecular insights of inhibition in sickle hemoglobin polymerization upon glutathionylation: hydrogen/deuterium exchange mass spectrometry and molecular dynamics simulation-based approach.

In sickle cell anemia, polymerization of hemoglobin in its deoxy state leads to the formation of insoluble fibers that result in sickling of red blood cells. Stereo-specific binding of isopropyl group of βVal6, the mutated amino-acid residue of a tetrameric sickle hemoglobin molecule (HbS), with hydrophobic groove of another HbS tetramer initiates the polymerization. Glutathionylation of βCys93 in HbS was reported to inhibit the polymerization. However, the mechanism of inhibition in polymerization is unknown to date. In our study, the molecular insights of inhibition in polymerization were investigated by monitoring the conformational dynamics in solution phase using hydrogen/deuterium exchange-based mass spectrometry. The conformational rigidity imparted due to glutathionylation of HbS results in solvent shielding of βVal6 and perturbation in the conformation of hydrophobic groove of HbS. Additionally, molecular dynamics simulation trajectory showed that the stereo-specific localization of glutathione moiety in the hydrophobic groove across the globin subunit interface of tetrameric HbS might contribute to inhibition in polymerization. These conformational insights in the inhibition of HbS polymerization upon glutathionylation might be translated in the molecularly targeted therapeutic approaches for sickle cell anemia.

Analysis of the Quaternary Structure of Hemoglobin Beckman Variant and Molecular Interpretation of Its Functional Abnormality: A Mass-Spectrometry-Based Approach.

Electrostatic attraction between α and β globin chains holds the subunits together in a tetrameric human hemoglobin molecule (α2 β2 ). Compared to normal globin chains, the affinity of a mutant chain to its partner globin might be different in genetic variants of hemoglobin. This leads to an unequal abundance of normal and variant hemoglobin in heterozygous samples, even though the rates of synthesis of both the normal and variant chains are the same. The aforementioned affinities across various globin chains might be assessed by quantification of the different forms of the tetramers present in a variant hemoglobin sample. In the present study, by exploiting mass differences between globin chains, differently populated hemoglobin tetramers present in hemoglobin (Hb) Beckman, a β variant (βA135D), were structurally characterized. The relative populations of dissymmetric tetramers (α2 β2 , α2 ββV , and α2 βV2 ) indicated that both β and βV have different affinities towards the α globin chain. Conformational dynamics analyzed from hydrogen/deuterium exchange kinetics of the three peptide fragments of Hb Beckman in its oxy state displayed molecular insight into its functional abnormality. However, in comparison to normal hemoglobin (α2 β2 ), the point mutation did not show any change in the collision cross-sections of the functionally active conformers of the variant hemoglobin molecules (α2 ββV and α2 βV2 ).

Immunodetection of Porcine Red Blood Cell Containing Food Ingredients Using a Porcine-Hemoglobin-Specific Monoclonal Antibody.

Monoclonal antibody (mAb) 24C12-E7 has been found to bind to a 12 kDa antigenic protein in the red blood cell (RBC) of porcine blood. The purpose of this study was to determine the identity of this 12 kDa protein and consequently examine its potential as a marker for monitoring porcine RBC-containing ingredients (PRBCIs) in foods. Proteomic techniques identified the 12 kDa antigenic protein to be a monomer of the tetrameric hemoglobin molecule. Further heat-processing of spray-dried PRBCIs diminishes its detectability. Whereas mAb 24C12-E7-based indirect enzyme-linked immunosorbent assay (iELISA) could detect 1% (v/v) or less of PRBCIs in raw and cooked ground meats (beef, pork and chicken), the detection limits were 3 to 30 times higher for spiked cooked beef and pork. The assay is effective for monitoring the presence of PRBCIs in foods to protect the billions of people that avoid consuming blood. In situations where these PRBCIs are present as ingredients in foods that have undergone further heat processing, the assay, however, may not be as sensitive depending on the types of sample matrix, types of PRBCIs and the level of inclusion.

Impact of polymer geometry on the interactions of protein-PEG conjugates

The conjugation of high molecular weight polyethylene glycol (PEG) to an active pharmaceutical ingredient (API) is an attractive strategy for the modification of biophysical and biodistribution properties of the API. Indeed, several therapeutic proteins conjugated to PEG have been safely administered in the clinic. While there have been studies on the configuration of these conjugates in solution, investigations on the impact of PEG geometry on protein-PEG conjugate interactions is limited. In this study, we use dynamic light scattering (DLS), rheology, and small-angle neutron scattering (SANS) to investigate the biophysical solution and interaction behavior of a 50kDa Fab protein attached to either a linear or tetrameric (branched) 40kDa PEG molecule. The hydrodynamic radii, diffusivity, viscosity and pair distance distribution function (PDDF) were obtained for the protein-PEG conjugates in solution. An analysis revealed that interactions between unconjugated proteins were quite attractive, however linear PEG-protein conjugates exhibited net repulsive interactions, similar to that of the unconjugated polymer. Tetramer PEG-protein conjugates on the other hand, exhibited a net weak attractive interaction, indicating a more balanced distribution of repulsive and attractive interaction states. Further analysis of the SANS data using geometric models consistent with the PDDF elucidated the conjugates' equilibrium configuration in solution. Insights gained from measurements and analysis used here can also be useful in predicting how conjugate geometries affect viscosity and aggregation behavior, which are important in determining suitable protein-polymer drug formulations.

Antibacterial Synthetic Peptides Derived from Bovine Lactoferricin Exhibit Cytotoxic Effect against MDA-MB-468 and MDA-MB-231 Breast Cancer Cell Lines.

Linear, dimeric, tetrameric, and cyclic peptides derived from lactoferricin B, containing the RRWQWR motif, were designed, synthesized, purified, and characterized using RP-HPLC chromatography and MALDI-TOF mass spectrometry. The antibacterial activity of the designed peptides against E. coli (ATCC 11775 and 25922) and their cytotoxic effect against MDA-MB-468 and MDA-MB-231 breast cancer cell lines were evaluated. Dimeric and tetrameric peptides showed higher antibacterial activity in both bacteria strains than linear peptides. The dimeric peptide (RRWQWR)2K-Ahx exhibited the highest antibacterial activity against the tested bacterial strains. Furthermore, the peptides with high antibacterial activity exhibited significant cytotoxic effect against the tested breast cancer cell lines. This cytotoxic effect was fast and dependent on the peptide concentration. The tetrameric molecule containing RRWQWR motif has an optimal cytotoxic effect at a concentration of 22 µM. The evaluated dimeric and tetrameric peptides could be considered as candidates for developing new therapeutic agents against breast cancer. Polyvalence of linear sequences could be considered as a novel and versatile strategy for obtaining molecules with high anticancer activity.

Adsorption Induced Changes of Human Hemoglobin on Ferric Pyrophosphate Nanoparticle Surface Probed by Isotope Exchange Mass Spectrometry: An Implication on Structure-Function Correlation.

In general, proteins in the biological system interact with nanoparticles (NPs) via adsorption on the particle surface. Understanding the adsorption at the molecular level is crucial to explore NP-protein interactions. The increasing concerns about the risk to human health on NP exposure have been explored through the discovery of a handful protein biomarkers and biochemical analysis. However, detailed information on structural perturbation and associated functional changes of proteins on interaction with NPs is limited. Erythrocytes (red blood cells) are devoid of defense mechanism of protecting NP penetration through endocytosis. Therefore, it is important to investigate the interaction of erythrocyte proteins with NPs. Hemoglobin, the most abundant protein of human erythrocyte, is a tetrameric molecule consisting of α- and β-globin chains in duplicate. In the present study, we have used hemoglobin as a model system to investigate NP-protein interaction with ferric pyrophosphate NPs [NP-Fe4(P2O7)3]. We report the formation of a bioconjugate of hemoglobin upon adsorption to NP-Fe4(P2O7)3 surface. Analysis of the bioconjugate indicated that Fe3+ ion of NP-Fe4(P2O7)3 contributed in the bioconjugate formation. Using hydrogen/deuterium exchange based mass spectrometry, it was observed that the amino termini of α- and β-globin chains of hemoglobin were involved in the adsorption on NP surface whereas the carboxy termini of both chains became more flexible in its conformation compared to the respective regions of the normal hemoglobin. Circular dichroism spectra of desorbed hemoglobin indicated an adsorption induced localized structural change in the protein molecule. The formation of bioconjugate led to functional alteration of hemoglobin, as probed by oxygen binding assay. Thus, we hypothesize that the large amount of energy released upon adsorption of hemoglobin to NP surface might be the fundamental cause of structural perturbation of human hemoglobin and subsequent formation of the bioconjugate with an altered function.

An unusual coordination of a 4-azopyrazol-5-one heterocyclic derivative with metals. Synthesis, X-ray studies, spectroscopic characteristics, and theoretical modeling

A series of novel transition and rare-earth metal complexes of (Z)-4-((1-(benzo[d]thiazol-2-yl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)diazenyl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (HL) were isolated and identified by FT IR, EPR, 1H NMR, and UV–VIS spectroscopy. The crystallographic data for [Cu2Cl3(L)(H2O)]2·1.5H2O·CH2Cl2 were obtained. The compound exists in the form of a tetranuclear species with two different metal coordination modes. The coordination polyhedra of two cations can be described as somewhat distorted tetragonal pyramids as well as two other Cu atoms of the tetrameric molecule adopt the trigonal bipyramidal coordination. The bridging chloride ligands link differently coordinated metal cations. The quantum chemical calculations indicate the existence of a ferromagnetic exchange interaction in pairs. That is in agreement with the EPR spectrum of the substance. The complex formation constants in water-ethanol solutions were determined.

Application of isotope exchange based mass spectrometry to understand the mechanism of inhibition of sickle hemoglobin polymerization upon oxygenation.

Sickle hemoglobin (HbS) polymerization initiates in the deoxy state with the binding of hydrophobic patch formed by the isopropyl group of βVal6 residue of a hemoglobin tetramer with the hydrophobic pocket of another tetramer, whose hydrophobic patch binds to the hydrophobic groove of a third molecule. Subsequent elongation of a single stranded polymer followed by the formation of a double strand and finally combination of seven such pairs of double strands results in a fourteen stranded fibrous polymer. Precipitation of this fiber inside the erythrocytes results in sickling of red blood cells. Surprisingly, the polymerization does not occur in the oxy state of HbS. Due to the unavailability of crystal structure of oxy form of HbS, the molecular basis of inhibition of polymerization in the oxy state is unknown to date. In the present study, we have attempted to understand the molecular mechanism of inhibition of polymerization by exploiting the exchange of backbone amide hydrogens of HbS with deuterated solvent. Hydrogen/deuterium exchange kinetics of peptide amide hydrogens of both oxy and deoxy form of HbS were monitored through ESI mass spectrometry. Upon oxygenation changes in the conformational flexibility across different regions of α and β globin chains in the tetrameric HbS molecule were investigated. It was observed that oxygenation led to perturbation in the conformation of several residues around the hydrophobic patch, groove of a tetramer and axial, lateral contacts across the double strands that are involved in HbS polymerization.

Characterization of T Cells Specific for CFP-10 and ESAT-6 in Mycobacterium tuberculosis-Infected Mauritian Cynomolgus Macaques.

Nonhuman primates can be used to study host immune responses to Mycobacterium tuberculosis Mauritian cynomolgus macaques (MCMs) are a unique group of animals that have limited major histocompatibility complex (MHC) genetic diversity, such that MHC-identical animals can be infected with M. tuberculosis Two MCMs homozygous for the relatively common M1 MHC haplotype were bronchoscopically infected with 41 CFU of the M. tuberculosis Erdman strain. Four other MCMs, which had at least one copy of the M1 MHC haplotype, were infected with a lower dose of 3 CFU M. tuberculosis All animals mounted similar T-cell responses to CFP-10 and ESAT-6. Two epitopes in CFP-10 were characterized, and the MHC class II alleles restricting them were determined. A third epitope in CFP-10 was identified but exhibited promiscuous restriction. The CFP-10 and ESAT-6 antigenic regions targeted by T cells in MCMs were comparable to those seen in cases of human M. tuberculosis infection. Our data lay the foundation for generating tetrameric molecules to study epitope-specific CD4 T cells in M. tuberculosis-infected MCMs, which may guide future testing of tuberculosis vaccines in nonhuman primates.

Conformational Changes and Competitive Adsorption between Serum Albumin and Hemoglobin on Bioceramic Substrates.

Traditional methods to analyze interactions and conformational changes of proteins adsorbed onto biomaterials are limited by the protein's associations with the substrate material and the complexity of the surrounding media. We have used EPR spectroscopy in combination with site-directed spin labeling (SDSL) to investigate single protein and competitive adsorption kinetics of horse hemoglobin (Hgb) and bovine serum albumin (BSA) on a silica-calcium-phosphate bioceramic substrate. Combined continuous wave and pulsed (DEER) EPR techniques were employed to monitor local mobility/flexibility changes within the proteins and tertiary structure dynamics upon adsorption. An alternate labeling technique was introduced to allow for specific quantification of each protein adsorbed to the bioceramic surface. We show that at buffer pH 7.4 and 4.7 the amount of adsorbed hemoglobin was increased by a factor of 4-5 compared with BSA. The tertiary structure of hemoglobin was strongly affected upon adsorption, leading to a dissociation of the tetrameric molecule into monomers or αβ dimers. When the bioceramic substrate was previously functionalized with a layer of BSA, dissociation was reduced by 71 % compared with the untreated surface, indicating a "primer" effect of BSA for better adhesion of the globular hemoglobin.

Synthesis and characterization of sterically crowded aryloxides: ‘Mitsubishi’-class of tetrametallic aluminum complexes

A new series of tetrameric aluminum molecules with a Mitsubishi structure, composed of two cage-shaped triphenolic ligands with a tripod-chelated structure, have been synthesized and characterized. Transmetalation from the cage-shaped borates was a proper method for the synthesis of these aluminum complexes. All complexes were determined via X-ray crystallography and NMR measurements. These Lewis acidic aluminum complexes catalyze the hetero Diels Alder reaction of the Danishefsky diene with benzaldehyde.

Structure of the second Single Stranded DNA Binding protein (SSBb) from Mycobacterium smegmatis.

All mycobacteria with sequenced genomes, except M. leprae, have a second Single Stranded DNA Binding protein (SSBb) in addition to the canonical one (SSBa). This paralogue from M. smegmatis (MsSSBb) has been cloned, expressed and purified. The protein, which is probably involved in stress response, has been crystallized and X-ray analyzed in the first structure elucidation of a mycobacterial SSBb. In spite of the low sequence identity between SSBas and SSBbs in mycobacteria, the tertiary and quaternary structure of the DNA binding domain of MsSSBb is similar to that observed in mycobacterial SSBas. In particular, the quaternary structure is 'clamped' using a C-terminal stretch of the N-domain, which endows the tetrameric molecule with additional stability and its characteristic shape. Comparison involving available, rather limited, structural data on SSBbs from other sources, appears to suggest that SSBbs could exhibit higher structural variability than SSBas do.

Molecular clusters Cs3X3 and Cs4X4 (X = Br, I): Quantum chemical study of structure and thermodynamic properties

AbstractThe properties of trimer Cs3X3 and tetramer Cs4X4 (X = Br, I) molecules have been studied using DFT with B3LYP5 functional and MP2 and MP4 methods. Two equilibrium geometrical structures of trimers, hexagonal (D3 h) and “butterfly-shaped” (Cs), and one for tetramers, distorted cubic (Td), are confirmed to exist; geometrical parameters and vibrational spectra are determined. The relative concentration of Cs3X3 isomers has been evaluated; the butterfly-shaped isomer dominates over hexagonal in saturated vapour in a broad temperature range. The dissociation reactions through different channels have been considered and enthalpies of formation ΔfH°(0) of clusters determined:−858 ± 20 kJ mol−1 (Cs3Br3), −698 ± 20 kJ mol−1 (Cs3I3), −1270 ± 30 kJ mol−1 (Cs4Br4) and −1045 ± 30 kJ mol−1 (Cs4I4). The Gibbs free energies ∆rG°(T) calculated for the dissociation reactions of trimer and tetramer molecules have indicated that these molecules are resistive in narrow temperature range only and decompose spontaneous...

Crystal structure of WI4

Tungsten tetraiodide WI4 (1) is produced by a high-temperature reaction of WTe2 and I2 in a vacuum sealed ampoule. The crystals of 1 belong to the triclinic crystal symmetry, space group P-1, Z = 4, a = = 7.9291(3) A, b = 10.7695(4) A, c = 10.8117(4) A, α = 85.668(1)°, β = 71.772(1)°, γ = 71.559(1)°, V = = 831.60(5) A3, dcalc = 5.523 g/cm3. The structure of 1 consists of tetrameric W4I16 molecules in which W atoms are in a distorted octahedral environment formed by I atoms.

Regulation of Lipid Specific and Vitamin Specific Non-MHC Restricted T Cells by Antigen Presenting Cells and Their Therapeutic Potentials.

Since initial reports, more than 25 years ago, that T cells recognize lipids in the context on non-polymorphic CD1 molecules, our understanding of antigen presentation to non-peptide-specific T cell populations has deepened. It is now clear that αβ T cells bearing semi-invariant T cell receptor, as well as subsets of γδ T cells, recognize a variety of self and non-self lipids and contribute to shaping immune responses via cross talk with dendritic cells and B cells. Furthermore, it has been demonstrated that small molecules derived from the microbial riboflavin biosynthetic pathway (vitamin B2) bind monomorphic MR1 molecules and activate mucosal-associated invariant T cells, another population of semi-invariant T cells. Novel insights in the biological relevance of non-peptide-specific T cells have emerged with the development of tetrameric CD1 and MR1 molecules, which has allowed accurate enumeration and functional analysis of CD1- and MR1-restricted T cells in humans and discovery of novel populations of semi-invariant T cells. The phenotype and function of non-peptide-specific T cells will be discussed in the context of the known distribution of CD1 and MR1 molecules by different subsets of antigen-presenting cells at steady state and following infection. Concurrent modulation of CD1 transcription and lipid biosynthetic pathways upon TLR stimulation, coupled with efficient lipid antigen processing, result in the increased cell surface expression of antigenic CD1–lipid complexes. Similarly, MR1 expression is almost undetectable in resting APC and it is upregulated following bacterial infection, likely due to stabilization of MR1 molecules by microbial antigens. The tight regulation of CD1 and MR1 expression at steady state and during infection may represent an important mechanism to limit autoreactivity, while promoting T cell responses to foreign antigens.

Is Single-4-Ring the Most Basic but Elusive Secondary Building Unit That Transforms to Larger Structures in Zinc Phosphate Chemistry?

Haloaryl phosphates (X-dippH2, X = Cl, Br, I) react with zinc acetate in the presence of collidine or 2-aminopyridine (2-apy) to yield zinc phosphate clusters [Zn(X-dipp)(collidine)]4 (X = Cl (1), Br (2), I (3)) and [Zn(X-dipp)(2-apy)]4·2MeOH (X = Cl (4), Br (5), I (6)), respectively. Single-crystal X-ray diffraction studies reveal that collidine and 2-apy capped zinc phosphates 1-6 exist as discrete tetrameric zinc phosphate molecules, exhibiting a cubane-shaped D4R core. In contrast, when the same reaction has been carried out in the presence of 4-cyanopyridine (4-CNpy), polymeric zinc phosphates {[Zn4(X-dipp)4(4-CNpy)2(MeOH)2]·2H2O}n (X = Cl (7), Br (8), I (9)) have been isolated. Compounds 7-9 are square-wave-shaped, one-dimensional polymers composed of fused S4R repeating units. The common structural motif found both in D4R cubanes 1-6 and polymers 7-9 is the S4R building block, which presumably undergoes further fusion because of the coordinative unsaturation at zinc and the simultaneous presence of free P═O. The closed shell cubanes 1-6 are obviously formed by a face-to-face dimerization involving two S4R units in which the two P═O groups are in cis-configuration. On the other hand, the one-dimensional (1-D) square-wave polymers 7-9 are formed from a face-to-face association of S4R building units in which the two P═O groups are in a trans-configuration. In order to stabilize these elusive S4R zinc phosphates, the reaction between Cl-dippH2 and zinc acetate was carried out in the presence of excess imidazole as an ancillary ligand (1:1:4), although only an imidazole decorated cubane cluster [Zn(Cl-dipp)(imz)]4.2MeOH (10) was isolated. The chelating N,N'-donor 1,10-phenanthroline ligand was used to eventually isolate cyclic S4R phosphate [Zn(μ2-Cl-dipp)(1,10-phen)(OH2)]2·MeOH·H2O (11). The change of Zn(2+) source to zinc nitrate and the phosphate source to 2,6-dimethylphenyl phosphate (dmppH2) led to the isolation of another polymeric phosphate [Zn(dmpp)(MeOH)]n (12), with a zigzag backbone, formed through an edge-to-edge to polymerization of S4R building units with P═O groups in trans-configuration. The isolation of four different structural types of zinc phosphates A-D in the present study can be rationalized in terms of fusion of S4R rings in a variety of ways to either produce discrete clusters or 1-D polymers.

A Monte Carlo Method for Simulating Drying Temperature of the Gas-Sensing Material Based on Polyacrylonitrile

The calculations of the dimer, trimer and tetramer molecules of polyacrylonitrile (PAN) using the quantum-chemical method are carried out. The computer simulation of formation of a polymer PAN chain by the Monte-Carlo method taken with the Metropolis and Wang-Landau algorithm is done. Technology of fabrication of gas-sensing material based on metal-containing PAN is developed. Electroconductive metal-containing PAN films are fabricated by method of pyrolysis under the influence of incoherent IR-radiation. Their sensing properties are studied.

Importance of the monomer-dimer-tetramer interconversion of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.

The molecular machinery of the cyanobacterial circadian clock oscillator consists of three proteins, KaiA, KaiB and KaiC, which interact with each other to generate circadian oscillations in the presence of ATP (the in vitro KaiABC clock oscillator). KaiB comprises four subunits organized as a dimer of dimers. Our previous study suggested that, on interaction with KaiC, the tetrameric KaiB molecule dissociates into two molecules of dimeric KaiB. It is uncertain whether KaiB also exists as a monomer and whether the KaiB monomer can drive normal circadian oscillation. To address these questions, we constructed a new KaiB oligomer mutant with an N-terminal deletion, KaiB10-108 . KaiB10-108 was a monomer at 4°C but a dimer at 35°C. KaiB10-108 was able to drive normal clock oscillation in an in vitro reconstituted KaiABC clock oscillator at 25°C, but it was not able to drive normal circadian gene expression rhythms in cyanobacterial cells at 41°C. Wild-type KaiB existed in equilibrium between a dimer and tetramer at lower KaiB concentrations or in the presence of 1m NaCl. Our findings suggest that KaiB is in equilibrium between a monomer, dimer and tetramer in cyanobacterial cells.