Mixture Of Trimers Research Articles

Functional characterization of the different oligomeric forms of human surfactant protein SP-D

Surfactant Protein D (SP-D) is a collectin protein that participates in the innate immune defense of the lungs. SP-D mediates the clearance of invading microorganisms by opsonization, aggregation or direct killing, which are lately removed by macrophages.SP-D is found as a mixture of trimers, hexamers, dodecamers and higher order oligomers, “fuzzy balls”. However, it is unknown whether there are differences between these oligomeric forms in functions, activity or potency.In the present work, we have obtained fractions enriched in trimers, hexamers and fuzzy balls of full-length recombinant human (rh) SP-D by size exclusion chromatography, in a sufficient amount to perform functional assays. We have evaluated the differences in protein lectin-dependent activity relative to aggregation and binding to E. coli, one of the ligands of SP-D in vivo. Fuzzy balls are the most active oligomeric form in terms of binding and aggregation of bacteria, achieving 2-fold binding higher than hexamers and 50% bacteria aggregation at very short times. Hexamers, recently described as a defined oligomeric form of the protein, have never been isolated or tested in terms of protein activity. rhSP-D hexamers efficiently bind and aggregate bacteria, achieving 50–60% aggregation at final time point and high protein concentrations. Nevertheless, trimers are not able to aggregate bacteria, although they bind to them. Therefore, SP-D potency, in functions that relay on the C-lectin activity of the protein, is proportional to the oligomeric state of the protein.

Photo-induced guest–host interactions produce grain boundaries between smectic blocks

A binary mixture of trimer I (20 wt%) and a host LC (80 wt%) exhibits grain boundaries consisting of nematic liquid crystals between smectic C blocks during a cooling process under UV irradiation.

Structural controlled pure metallo-triangular assembly through bisterpyridinyl Dibenzo[b,d]thiophene, Dibenzo[b,d]furan and Dibenzo[b,d]carbazole

A novel family of metallocycles was constructed by a one-pot self-assembly of three analogous bis(terpyridine) ligand monomers L1-L3, having different bent angles, with metal ions (Zn2+ or Cd2+). The dibenzo[b,d]thiophene-containing ligand L3 assembled with the metal ions to form a single trimer, whereas the dibenzo[b,d]furan-containing ligand L2 and dibenzo[b,d]carbazole-containing ligand L1 formed a mixture of trimers and tetramers. Heteroatoms (N, O, S) significantly contributed to the molecular size of the assemblies, owing to the bent angle of the bis-terpyridines ligands.

Supramolecular Assembly of Human Pulmonary Surfactant Protein SP-D

Pulmonary surfactant protein D (SP-D) is a glycoprotein from the collectin family that is a component of the lung surfactant system. It exhibits host defense and immune regulatory functions in addition to contributing to the homeostasis of the surfactant pool within the alveolar airspaces. It is known that the SP-D monomer forms trimers, which further associate into higher-order oligomers. However, the pathway and the interactions involved in the assembly of SP-D oligomers are not clearly understood. In the current study, a recombinant form of full-length human SP-D (rhSP-D) has been qualitatively and quantitatively studied by atomic force microscopy (AFM) and electrophoresis, with the aim to understand the conformational diversity and the determinants defining the oligomerization of the protein. The rhSP-D preparation studied is a mixture of trimers, hexamers, dodecamers and higher-order oligomeric species, with dodecamers accounting for more than 50% of the protein by mass. Similar structures were also found in hSP-D obtained from proteinosis patients, with the largest fuzzy-ball-like oligomers being more abundant in these samples. The proportion of dodecamer is increased under acidic conditions, accompanied by a conformational change into more compact configurations. Two hexamers appear to be the minimal necessary unit for dodecamer formation, with stabilization of the dodecamer occurring via non-covalent, ionic, and hydrophobic interactions between the individual N-terminal domains and the proximal area of the SP-D collagen stems.

Erratum to: Electrodeposition of 4,4′-di-tert-butylbiphenyl peroxide from the anodic oxidation of p-tert-butylphenol in an alkaline acetonitrile solution

The electrogeneration of pure 4,4′-di-tert-butylbiphenyl peroxide as an electrodeposit could be achieved on a platinum electrode through the anodic oxidation of p-tert butylphenol in the presence of Lutidine, in addition to several soluble oligomers. The electrochemical and theoretical studies have shown that the favored coupling reaction corresponds to the O–O bond, albeit it is generally being considered unstable. Without the strong base, a mixture of trimer, tetramer and pentamer was deposited. The peroxide product presents a different electroactivity to that of the phenolic compound, with the appearance of a redox process involving cathodic and anodic symmetrical peaks at −0.10/0.15 V versus SCE, which indicates the preference of the polymer to adhere onto the electrode surface. The thermal degradation has also been analyzed. Quantum-chemical calculations reveal the reason for the oxygen–oxygen coupling.

Electrodeposition of 4,4′-di-tert-butylbiphenyl peroxide from the anodic oxidation of p-tert-butylphenol in an alkaline acetonitrile solution

The electrogeneration of pure 4,4′-di-tert-butylbiphenyl peroxide as an electrodeposit could be achieved on a platinum electrode through the anodic oxidation of p-tert butylphenol in the presence of Lutidine, in addition to several soluble oligomers. The electrochemical and theoretical studies have shown that the favored coupling reaction corresponds to the O–O bond, albeit it is generally being considered unstable. Without the strong base, a mixture of trimer, tetramer and pentamer was deposited. The peroxide product presents a different electroactivity to that of the phenolic compound, with the appearance of a redox process involving cathodic and anodic symmetrical peaks at −0.10/0.15 V versus SCE, which indicates the preference of the polymer to adhere onto the electrode surface. The thermal degradation has also been analyzed. Quantum-chemical calculations reveal the reason for the oxygen–oxygen coupling.

Structural Characterization of Human Pulmonary Surfactant Protein SP-D by Atomic Force Microscopy

Pulmonary surfactant protein D (SP-D), a C-type (Ca2+-dependent) lectin, belongs to the collectin family. It is found mainly in lung surfactant, participating in the innate immune defense of the lungs.SP-D monomer contains four structural domains: an N-terminus domain, a collagen region, an α-helical coiled-coil neck and a C-terminus carbohydrate recognition domain (CRD). Monomers form trimers through folding of the collagenous region into triple helices and the assembly of a coiled-coil bundle of α-helices in the neck region. These trimers are stabilized by two disulfide bonds in the cysteine-rich N-terminal domain. Trimers associate into higher order oligomers whose size and conformation is sensitive to environmental factors and the conditions during purification and storage. Despite extensive studies carried out to characterize the oligomerization process of SP-D, the pathway and type of interactions involved in the formation of large oligomers, are not clearly understood. In the current study, a structural characterization of recombinant human SP-D (rhSP-D), produced in mammalian CHO cells, has been carried out by Atomic Force Microscopy (AFM) and electrophoresis. The goal has been the determination of the distribution of oligomeric forms, the exploration of the possible oligomerization pathway and the description of the conformational diversity of rhSP-D. AFM experiments revealed that rhSP-D is a mixture of trimers, hexamers, dodecamers, and larger oligomeric species identified as “fuzzy balls”, with the most abundant structure being the dodecamer under the conditions of these experiments. We have developed a cross-linking protocol to detect the presence of SP-D dodecameric forms by PAGE-SDS, in which dodecamer is only visualized after chemical crosslinking and in the presence of denaturing agents, indicating the importance of hydrophobic interactions in dodecamer formation.

Insights into the aggregation mechanism of Aβ(25–40)

The hydrophobic fragment of the Alzheimer's related β-amyloid (Aβ) peptide, Aβ(25–40), aggregates and forms insoluble amyloid fibrils at a rate similar to the full-length peptide. In order to gain insight into the fibrillization of Aβ(25–40) and the ability of the flavonoid myricetin to inhibit its aggregation, the isoleucine at position 32 (I32A) and the glycine at position 37 (G37A) in the full-length peptide were replaced with alanine. Thioflavin T assays indicate that substitution of isoleucine for alanine significantly reduces the rate and extent of fibrillization compared to the Aβ(25–40) and G37A peptides. Although all three peptides are fully disordered initially, circular dichroism studies suggest the structure of the I32A and G37A peptides are different from the parent peptide Aβ(25–40). Introduction of myricetin to the peptide samples results in modest structural changes for the Aβ(25–40) and G37A peptides but not the I32A peptide. Aβ(25–40) oligomers were predominantly tetramers, whereas I32A and G37A oligomers were a mixture of trimers and dimers. After 48h of incubation at 37°C, the amount of tetramers and trimers in solution dropped for the Aβ(25–40) and G37A peptides but remained similar for the I32A peptide. Incubation of Aβ(25–40) with myricetin increased the relative proportion of trimers to tetramers. Ultraviolet resonance Raman studies suggests that the I32A peptide may be more hydrated than the Aβ(25–40) and G37A peptides. Taken together, these data indicate the structural changes observed for the Aβ(25–40) and G37A peptides upon introduction of myricetin are localized around residue 32 and could arise from hydrophobic interactions between the peptide and the flavonoid or interference with the self-association of the peptide in this region. Substitution of isoleucine at position 32 with alanine had little effect on the peptide's secondary structure but dramatically decreased the propensity of the peptide fibrillize.

Expression and characterization of recombinant chicken mannose binding lectin

Mannose binding lectin (MBL) is a serum collagenous C-type lectin that plays an important role in the innate immune protection against pathogens. Previously, human and mouse studies have demonstrated that MBL binds a broad range of pathogens that results in their neutralization through agglutination, enhanced phagocytosis, and/or complement activation via the lectin pathway. The role of MBL in chicken is not well understood although the MBL concentration in serum seems to correlate with protection against infections. To investigate the role of MBL in chicken further, recombinant chicken MBL (RcMBL) was produced in HeLa R19 cells and purified using mannan affinity chromatography followed by gel filtration. RcMBL was shown to be structurally and functionally similar to native chicken MBL (NcMBL) isolated from serum. RcMBL is expressed as an oligomeric protein (mixture of trimers and oligomerized trimers) with a monomeric mass of 26kDa as determined by mass spectrometry, corresponding to the predicted mass. Glycan array analysis indicated that RcMBL bound most strongly to high-mannose glycans but also glycans with terminal fucose and GlcNac residues. The biological activity of RcMBL was demonstrated via its capacity to agglutinate Salmonella Typhimurium and to inhibit the hemagglutination activity of influenza A virus. The production of a structurally well-characterized and functionally active RcMBL will facilitate detailed studies into the protective role of MBL in innate defense against pathogens in chicken and other avian species.

A multivalent clade C HIV-1 Env trimer cocktail elicits a higher magnitude of neutralizing antibodies than any individual component.

The sequence diversity of human immunodeficiency virus type 1 (HIV-1) presents a formidable challenge to the generation of an HIV-1 vaccine. One strategy to address such sequence diversity and to improve the magnitude of neutralizing antibodies (NAbs) is to utilize multivalent mixtures of HIV-1 envelope (Env) immunogens. Here we report the generation and characterization of three novel, acute clade C HIV-1 Env gp140 trimers (459C, 405C, and 939C), each with unique antigenic properties. Among the single trimers tested, 459C elicited the most potent NAb responses in vaccinated guinea pigs. We evaluated the immunogenicity of various mixtures of clade C Env trimers and found that a quadrivalent cocktail of clade C trimers elicited a greater magnitude of NAbs against a panel of tier 1A and 1B viruses than any single clade C trimer alone, demonstrating that the mixture had an advantage over all individual components of the cocktail. These data suggest that vaccination with a mixture of clade C Env trimers represents a promising strategy to augment vaccine-elicited NAb responses. It is currently not known how to generate potent NAbs to the diverse circulating HIV-1 Envs by vaccination. One strategy to address this diversity is to utilize mixtures of different soluble HIV-1 envelope proteins. In this study, we generated and characterized three distinct, novel, acute clade C soluble trimers. We vaccinated guinea pigs with single trimers as well as mixtures of trimers, and we found that a mixture of four trimers elicited a greater magnitude of NAbs than any single trimer within the mixture. The results of this study suggest that further development of Env trimer cocktails is warranted.

Getting CAD in Shape: The Atomic Structure of Human Dihydroorotase Domain

CAD is a large multifunctional polypeptide that initiates and controls the de novo biosynthesis of pyrimidines in animals. In this issue of Structure, Grande-García and colleagues provide the first atomic information of this antitumoral target by reporting the crystal structure of the dihydroorotase domain of human CAD.

Production of multivalent protein binders using a self‐trimerizing collagen‐like peptide scaffold

A class of multivalent protein binders was designed to overcome the limitations of low-affinity therapeutic antibodies. These binders, termed "collabodies," use a triplex-forming collagen-like peptide to drive the trimerization of a heterologous target-binding domain. Different forms of collabody, consisting of the human single-chain variable fragment (scFv) fused to either the N or C terminus of the collagen-like peptide scaffold (Gly-Pro-Pro)(10), were stably expressed as soluble secretory proteins in mammalian cells. The collabody consisting of scFv fused to the N terminus of collagen scaffold is present as a homotrimer, whereas it exhibited a mixture of trimer and interchain disulfide-bonded hexamer when cysteine residues were introduced and flanked the scaffold. The collagenous motif in collabody is prolyl-hydroxylated, with remarkable thermal and serum stabilities. The collabody erb_scFv-Col bound to the extracellular domain of epidermal growth factor receptor with a binding strength approximately 20- and 1000-fold stronger than the bivalent and monovalent counterparts, respectively. The trimeric collagen scaffold does not compromise the functionality of the binding moieties of parental immunoglobulin G (IgG); therefore, it could be applied to fuse other protein molecules to acquire significantly improved targeting-binding strengths.

Multiple functions of the von Willebrand Factor A domain in matrilins: secretion, assembly, and proteolysis

The von Willebrand Factor A (vWF A) domain is one of the most widely distributed structural modules in cell-matrix adhesive molecules such as intergrins and extracellular matrix proteins. Mutations in the vWF A domain of matrilin-3 cause multiple epiphyseal dysplasia (MED), however the pathological mechanism remains to be determined. Previously we showed that the vWF A domain in matrilin-1 mediates formation of a filamentous matrix network through metal-ion dependent adhesion sites in the domain. Here we show two new functions of the vWF A domain in cartilage-specific matrilins (1 and 3). First, vWF A domain regulates oligomerization of matrilins. Insertion of a vWF A domain into matrilin-3 converts the formation of a mixture of matrilin-3 tetramer, trimer, and dimer into a tetramer only, while deletion of a vWF A domain from matrilin-1 converts the formation of the native matrilin-1 trimer into a mixture of trimer and dimer. Second, the vWF A domain protects matrilin-1 from proteolysis. We identified a latent proteolytic site next to the vWF A2 domain in matrilin-1, which is sensitive to the inhibitors of matrix proteases. Deletion of the abutting vWF A domain results in degradation of matrilin-1, presumably by exposing the adjacent proteolytic site. In addition, we also confirmed the vWF A domain is vital for the secretion of matrilin-3. Secretion of the mutant matrilin-3 harbouring a point mutation within the vWF A domain, as occurred in MED patients, is markedly reduced and delayed, resulting from intracellular retention of the mutant matrilin-3. Taken together, our data suggest that different mutations/deletions of the vWF A domain in matrilins may lead to distinct pathological mechanisms due to the multiple functions of the vWF A domain.

Kinetics and mechanism of the reaction between 2,3,4,5,6-pentafluorophenylacetonitrile and guanidine-like bases and the structure of the products

The reactions between the 2,3,4,5,6-pentafluorophenylacetonitrile (PFPA) and such strong N-bases as 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD) and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) have been studied by UV–vis kinetic, high-resolution LSIMS, 19F NMR and FT-IR methods. The products of these reactions (compounds 1 and 2 and oligomers) have been isolated and their structures have been studied using the methods mentioned. They have been identified as ortho or para substituted dimers and a mixture of trimers, tetramers and pentamers of PFPA. All the products are formed in a relatively slow processes described by various low equilibrium constants. The structures of the ortho or para substituted dimers have been visualized by PM5 semiempirical calculations.

Processing, Stability, and Receptor Binding Properties of Oligomeric Envelope Glycoprotein from a Primary HIV-1 Isolate

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is thought to exist on the virion surface as a trimer of non-covalently associated gp120/gp41 molecules. We expressed trimeric envelope glycoprotein from three primary, macrophage tropic HIV-1 isolates in baby hamster kidney cells and analyzed the furin-mediated cleavage, stability, and receptor binding properties of the oligomers. The envelope glycoprotein was secreted in a soluble form deleted of its transmembrane anchor and the intracytoplasmic domain (gp140). A mixture of trimers, dimers, and monomers of gp140 as well as monomeric gp120 was detected on polyacrylamide gels. Analysis by sucrose gradient centrifugation revealed that trimers and dimers were essentially composed of uncleaved gp140, whereas most of the gp120 was found in the monomeric fraction. To analyze the effect of the cleavage of gp140 to gp120/Delta41 on trimerization, we co-expressed the furin protease along with gp140. Surprisingly, furin expression changed the subcellular localization of the envelope glycoprotein, which became in majority sequestered in the major furin compartment, the trans-Golgi network, as judged by confocal laser microscopy. The envelope glycoprotein secreted from furin-co-expressing cells was almost completely cleaved to gp120 and Deltagp41, but gp120 was found exclusively in the monomeric fraction, with a few residual oligomers being composed of uncleaved gp140. Secreted uncleaved gp140 trimers were purified to homogeneity and analyzed for their capacity to interact with cellular receptors CD4 and CC chemokine receptor 5 (CCR5). Receptor binding was analyzed on CD4- and CCR5-expressing cells as well as on peripheral blood mononuclear cells. Trimers showed greatly reduced binding to CD4 as compared with monomers. Neither monomers nor trimers bound directly to CCR5. In conclusion, our results show that the cleaved form of the envelope glycoprotein does not form stable trimers, suggesting that gp120/gp41 oligomers on the virion surface might be stabilized by a yet to be identified mechanism and that the virion might attach to CD4 via a monomeric form of gp120. These results are relevant to the development of an envelope-based vaccine against AIDS.

Thermostability, oligomerization and DNA-binding properties of the regulatory protein ArgR from the hyperthermophilic bacterium Thermotoga neapolitana.

The hexameric regulatory protein ArgR formed by arginine-mediated dimerization of identical trimers governs the expression of genes required for arginine metabolism and some other genes in mesophilic and moderately thermophilic bacteria. We have cloned the argR gene from two hyperthermophilic bacteria of the genus Thermotoga. The two-domain ArgR proteins encoded by T. neapolitana and T. maritima share a low degree of sequence similarity with other bacterial arginine repressors. The ArgR protein from T. neapolitana binds to an operator located just upstream of its coding sequence and, therefore, the argR gene may be autoregulated. The protein has extremely high intrinsic thermostability and tolerance to urea. Moreover, its binding to target DNA increases the melting temperature by approximately 15 degrees C. The formation of oligomeric ArgR-DNA complexes is a function of protein concentration, with hexameric complexes being favoured at higher concentrations. In the presence of arginine the hyperthermophilic ArgR protein binds to its own operator, argRo, only by forming hexamer ArgR-DNA complexes, whereas both trimer-DNA and hexamer-DNA complexes are detected in the absence of arginine. However, the affinity of T. neapolitana ArgR for DNA has been found to be higher for a mixture of trimers and non-bound hexamers than for arginine-bound hexamers. Our data indicate that genes for arginine biosynthesis are clustered in a putative operon, which could also be regulated by the ArgR protein, in the hyperthermophilic host.

A computational study of structure, bonding, and aggregation of N–Lithioaziridine

Semiempirical, ab initio, and density functional methods were used to determine the aggregation states of unsolvated and solvated N–lithioaziridine. Among the aggregated forms studied, the computational results suggest that in the absence of solvating ligands, N–lithioaziridine will exist primarily as a mixture of trimers and tetramers. The THF solvated lithioaziridines favored a tetrameric state whereas the HMPA and TMEDA solvated forms favored lower aggregation states.

Infrared reflection absorption spectra of trimethylaluminum and dimethylaluminum hydride condensed layers on an SiO 2 surface with a buried metal layer substrate

Infrared reflection absorption spectroscopy (IRAS) of trimethylaluminum (TMA) and dimethylaluminum hydride (DMAH) layers condensed on an SiO 2 surface was carried out by using a buried metal layer (BML) substrate. TMA was a dimer in the experimental temperature range of 110 K < T < 160 K. The molecular orientation varied depending on the deposition temperature. The condensed layer of DMAH as-deposited at the temperature ranging from 110 to 130 K was a mixture of trimers and dimers. The dimers changed to trimers as the temperature increased, practically disappearing above 130 K. This temperature dependence was not reversible. The trimers did not change to dimers below 130 K. The ordered orientation of the trimer molecules was a stable DMAH condensed layer structure on the SiO 2 surface. The usefulness of BML-IRAS for studying the structure and molecular orientation of adsorbed layers on semiconductor materials is discussed.

Cyclo-oligomerization and rearrangement of some phenylated diynes by the RhCl 3Aliquat 336 and by the H 2PtCl 6Aliquat 336 catalysts under phase transfer conditions

The phenylated diynes 3,3′-oxybis(1-phenyl-1-propyne) ( 3), its thio-analog 4, 1,2-bis(phenylethynyl)benzene ( 5) and 1,8-bis(phenylethynyl)naphthalene ( 6) were reacted, under phase transfer conditions, in the presence of the ion pairs generated from Aliquat® 336 and either aqueous RhCl 3 or H 2PtCl 6. The nature of the products proved to depend on the catalyst employed. The rhodium catalyst promotes the conversion of 3 into a mixture of trimer 8 and dimer 7a, as well as the hydrolysis of the latter compound to give 1,3-dihydro-4,6,7-triphenyl-5-isobenzofuranmethanol ( 9). The sulfur compound 4 yields only hydrolysis-resistant 1,3-dihydro-5-[2-propynyl(thiomethyl)]benzo[ c]thiophen ( 7b), and 6 forms mainly dimer 17 accompanied by small amounts of 7-phenyl- and 7-chloro-12-phenylbenzo[ k]fluoranthene ( 16a and 16b, respectively). The platinum catalyst promotes particularly intramolecular cycloaddition reaction of the diynes. Compound 3 is transformed almost entirely to 1,3-dihydro-9-phenylindeno[1,2- c]pyran ( 12), 5 is isomerized to give solely 5-phenylindeno[1,2- a]indene ( 15), and 6 yields 16a and 12-phenylindino[2,1- a]phenalene ( 18), as the minor and major products.

Bis(ethyl‐3‐Oxo‐Butanolato‐O1,O3) ‐bis(2‐propanolato)titanium (tyzor) DCR) as a reactant and catalyst in the polymerization of 2‐isocyanatoethyl methacrylate (IEM)

AbstractThe reaction of bis(ethyl‐3‐oxo‐butanolato‐O1,O3)‐bis(2‐propanolato)titanium(Tyzor DCR and 2‐isocyanatoethyl methacrylate (IEM) in refluxing hexane produced a mixture of trimer and polymers of 2‐isocyanatoethyl methacrylate which varied considerably from 23 to 92% polymer as the molar ratio of bis(ethyl‐3‐oxo‐butanolato‐O1,O3)‐bis(2‐propanolato)titanium(Tyzor DCR) to 2‐isocyanatoethyl methacrylate (IEM) varied from 1:4 to 1:32. The IEM 1‐nylon polymer which was soluble in cresol and phenol was characterized by FTIR, 1H‐NMR, and 13C‐NMR. This analysis also showed that the methacrylate carbon–carbon double bond survived our reaction conditions intact. Based on atomic absorption determination of titanium, the molecular weight of the polymer ranged between 3000 and 4000 g/mol. Thermal gravimetric analysis (TGA) showed the polymer to be stable in air to approximately 250°C.