Evidence For Dimer Formation Research Articles

H2O2 oxidation of cysteine residues in c-Jun N-terminal kinase 2 (JNK2) contributes to redox regulation in human articular chondrocytes

Reactive oxygen species (ROS), in particular H2O2, regulate intracellular signaling through reversible oxidation of reactive protein thiols present in a number of kinases and phosphatases. H2O2 has been shown to regulate mitogen-activated protein kinase (MAPK) signaling depending on the cellular context. We report here that in human articular chondrocytes, the MAPK family member c-Jun N-terminal kinase 2 (JNK2) is activated by fibronectin fragments and low physiological levels of H2O2 and inhibited by oxidation due to elevated levels of H2O2 The kinase activity of affinity-purified, phosphorylated JNK2 from cultured chondrocytes was reversibly inhibited by 5-20 μm H2O2 Using dimedone-based chemical probes that react specifically with sulfenylated cysteines (RSOH), we identified Cys-222 in JNK2, a residue not conserved in JNK1 or JNK3, as a redox-reactive site. MS analysis of human recombinant JNK2 also detected further oxidation at Cys-222 and other cysteines to sulfinic (RSO2H) or sulfonic (RSO3H) acid. H2O2 treatment of JNK2 resulted in detectable levels of peptides containing intramolecular disulfides between Cys-222 and either Cys-213 or Cys-177, without evidence of dimer formation. Substitution of Cys-222 to alanine rendered JNK2 insensitive to H2O2 inhibition, unlike C177A and C213A variants. Two other JNK2 variants, C116A and C163A, were also resistant to oxidative inhibition. Cumulatively, these findings indicate differential regulation of JNK2 signaling dependent on H2O2 levels and point to key cysteine residues regulating JNK2 activity. As levels of intracellular H2O2 rise, a switch occurs from activation to inhibition of JNK2 activity, linking JNK2 regulation to the redox status of the cell.

Vibrational circular dichroism of a 2,5-diketopiperazine (DKP) peptide: Evidence for dimer formation in cyclo LL or LD diphenylalanine in the solid state.

The diastereomer diketopiperazine (DKP) peptides built on phenylalanine, namely, cyclo diphenylalanine LPhe-LPhe and LPhe-DPhe, were studied in the solid phase by vibrational circular dichroism (VCD) coupled to quantum chemical calculations. The unit structure of cyclo LPhe-LPhe in KBr pellets is a dimer bridged by two strong NH…O hydrogen bonds. The intense bisignate signature in the CO stretch region is interpreted in terms of two contributions arising from the free COs of the dimer and the antisymmetrical combination of the bound COs. In contrast, cyclo LPhe-DPhe shows no VCD signal in relation to its symmetric nature.

Structural and electrochemical characterization of metallo-porphyrins intercalated into ZnCr-layered double hydroxides: some evidence of dimer formation

In this study, inorganic–organic hybrids were prepared by intercalation of metal complexes (Zn and Fe) of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TSPP) into Zn2Cr layered double hydroxides (LDH) by coprecipitation method at constant pH. The resulting materials were characterized by powder X-ray diffraction, UV-Vis and FTIR spectroscopies showing the intercalation of porphyrins between the ZnCr layers. While monomer species are intercalated in the case of Zn2Cr-ZnTSPP, the intercalation of dimers is likely to occur in the case of Zn2Cr-FeTSPP. In both materials, a perpendicular orientation of the porphyrin ring against the hydroxide layers is proposed. Of particular interest is the good stability of ZnCr-LDH host over a wide pH range. The electrochemical behavior of these hybrid materials in both neutral (pH 7.0) and acid media (pH 4.5) was investigated by cyclic voltammetry. Zn2Cr-FeTSPP exhibits an electrocatalytic activity for the reduction of oxygen, hydrogen peroxide and nitrite.

Intermolecular Cross-Linking of Na+−Ca2+ Exchanger Proteins: Evidence for Dimer Formation

The cardiac Na (+)-Ca (2+) exchanger (NCX1) is modeled to contain nine transmembrane segments (TMS) with a pair of oppositely oriented, conserved sequences called the alpha-repeats that are important in ion transport. Residue 122 in the alpha-1 repeat is in proximity to residue 768 in TMS 6, and the two residues can be cross-linked . During studies on the substrate specificity of this intramolecular cross-link, we found evidence that NCX1 can form dimers. At 37 degrees C in the absence of extracellular Na (+), copper phenanthroline catalyzes disulfide bond formation between cysteines at position 122 in adjacent NCX1 proteins. Dimerization was confirmed by histidine tag pull-down experiments that demonstrate the association of untagged NCX1 with histidine-tagged NCX1. Dimerization occurs along a face of the protein that includes parts of the alpha-1 and alpha-2 repeats as well as parts of TMS 1 and TMS 2. We do not see cross-linking between residues in TMS 5, TMS 6, or TMS 7. These data provide the first evidence for dimer formation by the Na (+)-Ca (2+) exchanger.

Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ

The recently characterised 299-residue human XLF/Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4–DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1–233) homodimer at 2.3 Å resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex.

Structure and Regulatory Profile of the Monkeypox Inhibitor of Complement: Comparison to Homologs in Vaccinia and Variola and Evidence for Dimer Formation

The outbreak of monkeypox in the Unites States in the summer of 2003 was the first occurrence of this smallpox-like disease outside of Africa. This limited human epidemic resulted from cross-infection of prairie dogs by imported African rodents. Although there were no human fatalities, this outbreak illustrates that monkeypox is an emerging natural infection and a potential biological weapon. We characterized a virulence factor expressed by monkeypox (monkeypox inhibitor of complement enzymes or MOPICE). We also compared its structure and regulatory function to homologous complement regulatory proteins of variola (SPICE) and vaccinia (VCP). In multiple expression systems, 5-30% of MOPICE, SPICE, and VCP consisted of function-enhancing disulfide-linked homodimers. Mammalian cells infected with vaccinia virus also expressed VCP dimers. MOPICE bound human C3b/C4b intermediate to that of SPICE and VCP. Cofactor activity of MOPICE was similar to VCP, but both were approximately 100-fold less efficient than SPICE. SPICE and VCP, but not MOPICE, possessed decay-accelerating activity for the C3 and C5 convertases of the classical pathway. Additionally, all three regulators possessed heparin-binding capability. These studies demonstrate that MOPICE regulates human complement and suggest that dimerization is a prominent feature of these virulence factors. Thus, our data add novel information relative to the functional repertoire of these poxviral virulence factors. Furthermore, targeting and neutralizing these complement regulatory active sites via mAbs is a therapeutic approach that may enhance protection against smallpox.

Spectroscopic characterizations of the mixed Langmuir–Blodgett (LB) films of 2,2′-biquinoline molecules: Evidence of dimer formation

This communication reports the π– A isotherms and spectroscopic characterizations of mixed Langmuir and Langmuir–Blodgett (LB) films of non-amphiphilic 2,2′-biquinoline (BQ) molecules, mixed with polymethyl methacrylate (PMMA) and stearic acid (SA). The π– A isotherms and mole fraction vs. area per molecule studies indicate complete immiscibility of sample (BQ) and matrix (PMMA or SA) molecules. This immiscibility may lead to the formation of microcrystalline aggregates of BQ molecules in the mixed LB films. The scanning electron micrograph gives the visual evidence of microcrystalline aggregates of BQ molecules in the mixed LB films. UV–vis absorption, fluorescence and excitation spectroscopic studies reveal the nature of these microcrystalline aggregates. LB films lifted at higher surface pressure indicate the formation of dimer or higher order n-mers.

Evidence for dimer formation by an amphiphilic heptapeptide that mediates chloride and carboxyfluorescein release from liposomes

Heptapeptides having dioctadecyl, N-terminal hydrocarbon chains insert in phospholipid bilayer membranes and form pores through which at least chloride ions pass. Although amphiphilic, these compounds do not typically form vesicles themselves. They insert in the bilayers of phospholipid vesicles and mediate the release of carboxyfluorescein. Hill analysis indicates that at least two molecules of the amphiphile are involved in pore formation. In CD2Cl2, dimer formation is detected by NMR chemical shift changes. The anion release activity of individual anion transporters is increased by linking them covalently at the C-terminus or, even more, by linking them at the N-terminus. Evidence is presented that either linked molecule releases chloride from liposomes more effectively and rapidly than the individual transporter molecule at a comparable concentration.

Determination of pKa's for thymol blue in aqueous medium: evidence of dimer formation.

Formation constants for recrystallized thymol blue were determined in water, using the SQUAD and SUPERQUAD programs. The best model correlating spectrophotometric, potentiometric and conductimetric data was fitted with the dissociation of HL−=L2−+H+−log K=8.918±0.070 and H3L2−=2L2−+3H+−log K=29.806±0.133 with the SUPERQUAD program at variable low ionic strength (1.5×10−4–3.0×10−4 M); and HL=L2−+H+−log K=8.9±0.000, H3L2− =2L2−+3H+−log K=30.730±0.032, H4L2=2L2−+4H+−log K=32.106±0.033 with SQUAD at 1.1 M ionic strength.

Evidence of dimer formation in neat liquid 1,3,5-trifluorobenzene

1,3,5-Trifluorobenzene neat liquid has been studied in comparison with benzene and perfluorinated benzene using neutron diffraction experiments and molecular dynamics simulation. These investigations reveal that the short-range local order is markedly different in 1,3,5-trifluorobenzene liquid, and involves ‘dimers’, in which the molecules have a parallel stacked configuration, and an average intermolecular distance of less than 4 Å.

Pyrene excimer formation in Langmuir-Blodgett films

Molecular aggregate formation in Langmuir-Blodgett films of dye molecules has been investigated. Mixed layers of 1 -pyrenebutanoic acid (PHA) with cadmium stearate/stearic acid and pyrene with cadmium stearate/stearic acid have been deposited onto quartz substrates. Using the time-correlated single-photon counting technique and fluorescence spectroscopy, evidence for dimer formation in PHA multilayers and excimer formation in pyrene films was obtained. The different types of aggregates are attributed to structural differences between the pyrene and PHA mixed films.

New developments in spectroellipsometry: the challenge of surfaces

Advances in technology, especially semiconductor technology, are providing new opportunities for the development and application of spectroellipsometry and related techniques, particularly for real-time diagnostics and control. Two recent examples of surface and near-surface analysis illustrate possibilities. Reflectance-difference spectroscopy of (001) GaAs growth surfaces under ultra-high vacuum and atmospheric-pressure conditions has provided the first evidence of dimer formation during organometallic chemical vapor deposition (OMCVD). Kinetic-ellipsometric determination of the composition x of Al x Ga 1− x As alloys during chemical beam epitaxy (CBE), with x established by a history-independent, minimal-data approach, has been used to realize the first fully automatic closed-loop feedback system for controlling growth directly. Possible future directions are discussed.

Dimer formation on (001) GaAs under organometallic chemical vapor deposition growth conditions

We report the first evidence of dimer formation on semiconductor surfaces under atmospheric pressure (AP) conditions. We successfully employ reflectance-difference spectroscopy to link structures of well-defined (001) GaAs surfaces prepared in ultrahigh vacuum (UHV) by molecular-beam epitaxy with structures of (001) GaAs surfaces prepared in AP by organometallic chemical vapor deposition (OMCVD). These measurements reveal that surface reconstructions also occur under AP-OMCVD conditions, forming structures similar, possibly identical, to the (4×2), (2×4), c(4×4), and disordered c(4×4), that occur in UHV. Our results provide a justification for applying the results of UHV studies of these surfaces to non-UHV environments and yield new insights on the mechanisms and kinetics of non-UHV growth.

Photophysics at solid surfaces. Evidence of dimer formation and polarization of monomer and excimer fluorescences of pyrene in the adsorbed state on silica-gel surfaces

The fluorescence, fluorescence excitation and fluorescence-polarization spectra of pyrene adsorbed on a silica gel surface activated at 473 K have been observed as a function of the amount of adsorbed pyrene, as well as the ratio of fluorescence quantum yields of excimer-like fluorescence to monomer-like fluorescence. Kinetic considerations reveal that a part of the adsorbed pyrene forms a ground-state dimer. The excited dimer rearranges its conformation during the fluorescence lifetime from which excimer-like fluorescence is emitted. The degree of polarization, P, has values other than zero. These findings indicate that the excimer formation can be accomplished by slight displacement which does not depolarize the emission on the silicagel surface during the formation of the excimer from the ground-state dimer. It is also shown that the polarization of excimer emission is along the a axis of the pyrene molecule, and the excimer is favourably formed by b-axis excitation rather than a-axis excitation.

Aminoarsolanes—II. Conformational studies

1H and 13C NMR spectra of six dialkylaminoarsolanes were obtained as a function of solvent, concentration, and temperature. CI mass spectral data were also obtained as a function of retention time for the same compounds. These results suggest that the AsN bond is not labile as compared to the AsCl bond in chloroarsolanes. No evidence for dimer formation was noted either in solution or the gas phase under the investigated experimental conditions. The averaged solution conformation of the 1,3-dioxarsolane ring is discussed in terms of rapid intramolecular conformational averaging; nature of substituents at the 2,4, and 5 positions; and effects of temperature, solute concentration and solvent type.

Electron spin resonance studies of some copper(II) peptide complexes

Electron spin resonance studies have been made of a number of copper(II) peptides. For the copper(I) complexes of glycylglycine, diglycylglycine, and β-alanyl-L-histidine and its derivatives dimer formation takes place in frozen aqueous solutions. The application of computational procedures has enabled estimates of copper(II)–copper(II) separations to be made which are found to be compatible with structures resulting from X-ray measurements. No evidence for dimer formation at room temperature has emerged. For the 2:1 copper(II)L-cystinyl-bisglycinate complex the copper(II)–copper(II) separation is consistent with a folded conformation of the chelate.