Top-Down Mass Analysis of Protein Tyrosine Nitration: Comparison of Electron Capture Dissociation with “Slow-Heating” Tandem Mass Spectrometry Methods

Tyrosine nitration in proteins is an important post-translational modification (PTM) linked to various pathological conditions. When multiple potential sites of nitration exist, tandem mass spectrometry (MS/MS) methods provide unique tools to locate the nitro-tyrosine(s) precisely. Electron capture dissociation (ECD) is a powerful MS/MS method, different in its mechanisms to the “slow-heating” threshold fragmentation methods, such as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). Generally, ECD provides more homogeneous cleavage of the protein backbone and preserves labile PTMs. However recent studies in our laboratory demonstrated that ECD of doubly charged nitrated peptides is inhibited by the large electron affinity of the nitro group, while CID efficiency remains unaffected by nitration. Here, we have investigated the efficiency of ECD versus CID and IRMPD for top-down MS/MS analysis of multiply charged intact nitrated protein ions of myoglobin, lysozyme, and cytochrome c in a commercial Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. CID and IRMPD produced more cleavages in the vicinity of the sites of nitration than ECD. However the total number of ECD fragments was greater than those from CID or IRMPD, and many ECD fragments contained the site(s) of nitration. We conclude that ECD can be used in the top-down analysis of nitrated proteins, but precise localization of the sites of nitration may require either of the “slow-heating” methods

Isomeric effects in the gas-phase reactions of dichloroethene, C2H2Cl2, with a series of cations

A study of the reactions of a series of gas-phase cations (NH$_4^+$, H$_3$O$^+$, SF$_3^+$, CF$_3^+$, CF$^+$, SF5$^+$, SF$_2^+$, SF$^+$, CF$_2^+$, SF$_4^+$, O$_2^+$, Xe$^+$, N$_2$O$^+$, CO$_2^+$, Kr$^+$, CO$^+$, N$^+$, N$_2^+$, Ar$^+$, F$^+$ and Ne$^+$) with the three structural isomers of dichloroethene, i.e. 1,1-C$_2$H$_2$Cl$_2$, cis-1,2-C$_2$H$_2$Cl$_2$ and trans-1,2-C$_2$H$_2$Cl$_2$ is reported. The recombination energy of these ions spans the range 4.7-21.6 eV. Reaction rate coefficients and product branching ratios have been measured at 298 K in a selected ion flow tube. Collisional rate coefficients are calculated by modified average dipole orientation theory and compared with experimental data. Thermochemistry and mass balance have been used to predict the most feasible neutral products. Threshold photoelectron-photoion coincidence spectra have also been obtained for the three isomers of C$_2$H$_2$Cl$_2$ with photon energies in the range 10-23 eV. The fragment ion branching ratios have been compared with those of the flow tube study to determine the importance of long-range charge transfer. A strong influence of the isomeric structure of dichloroethene on the products of ion-molecule reactions has been observed for H$_3$O$^+$, CF$_3^+$, and CF$^+$. For 1,1-C$_2$H$_2$Cl$_2$ the reaction with H$_3$O$^+$ proceeds at the collisional rate with the only ionic product being 1,1-C$_2$H$_2$Cl$_2$H$^+$. However, the same reaction yields two more ionic products in the case of cis-1,2- and trans-1,2-C$_2$H$_2$Cl$_2$, but only proceeds with 14 % and 18 % efficiency, respectively. The CF$_3^+$ reaction proceeds with 56-80 % efficiency, the only ionic product for 1,1-C$_2$H$_2$Cl$_2$ being C$_2$H$_2$Cl$^+$ formed via Cl- abstraction, whereas the only ionic product for both 1,2-isomers is CHCl$_2^+$ corresponding to a breaking of the C=C double bond. Less profound isomeric effects, but still resulting in different products for 1,1- and 1,2-C$_2$H$_2$Cl$_2$ isomers, have been found in the reactions of SF$^+$, CO$_2^+$, CO$^+$, N$_2^+$, and Ar$^+$. Although these five ions have recombination energies above the ionization energy of any of the C$_2$H$_2$Cl$_2$ isomers and hence the threshold for long-range charge transfer, the results suggest that the formation of a collision complex at short range between these ions and C$_2$H$_2$Cl$_2$ is responsible for the observed effects

Binding of the baculovirus very late expression factor 1 (VLF-1) to different DNA structures

BACKGROUND: Baculovirus genomes encode a gene called very late expression factor 1 (VLF-1) that is a member of the integrase (Int) family of proteins. In this report we describe the binding properties of purified Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) VLF-1 to a number of different DNA structures including homologous regions. In addition, its enzymatic activity was examined. RESULTS: VLF-1 was expressed in a recombinant baculovirus as a fusion with both HA and HIS(6) tags and its binding activity to different DNA structures was tested. No binding was evident to single and double strand structures, very low binding was observed to Y-forks, more binding was observed to three-way junctions, whereas cruciform structures showed high levels of binding. VLF-1 binding was affected by divalent cations; optimal binding to three-way junctions and cruciforms was 2 and 0 mM MgCl(2), respectively. Homologous region (hr) sequences was also examined including oligomers designed to expose the hr palindrome as a hairpin, linear double strand, or H-shaped structure. Efficient binding was observed to the hairpin and H-shaped structure. No topoisomerase or endonuclease activity was detected. Sedimentation analysis indicated that *VLF-1 is present as a monomer. CONCLUSIONS: An HA- and HIS-tagged version of AcMNPV VLF-1 showed structure-dependent binding to DNA substrates with the highest binding affinity to cruciform DNA. These results are consistent with the involvement of VLF-1 in the processing of branched DNA molecules at the late stages of viral genome replication. We were unable to detect enzymatic activity associated with these complexes

Notes on beta-deformations of the pure spinor superstring in AdS(5) x S(5)

We study the properties of the vertex operator for the beta-deformation of the superstring in AdS(5) x S(5) in the pure spinor formalism. We discuss the action of supersymmetry on the infinitesimal beta-deformation, the application of the homological perturbation theory, and the relation between the worldsheet description and the spacetime supergravity description.Comment: LaTeX, 74pp

Structural and functional analysis of the baculovirus single-stranded DNA-binding protein LEF-3

AbstractThe single-stranded DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus consists of 385 amino acid residues, forms oligomers, and promotes Mg2+-independent unwinding of DNA duplexes and annealing of complementary DNA strands. Partial proteolysis revealed that the DNA-binding domain of LEF-3 is located within a central region (residues 28 to 326) that is relatively resistant to proteolysis. In contrast, the N-terminus (27 residues) and C-terminal portion (59 residues) are not involved in interaction with DNA and are readily accessible to proteolytic digestion. Circular dichroism analyses showed that LEF-3 is a folded protein with an estimated α-helix content of more than 40%, but it is structurally unstable and undergoes unfolding in aqueous solutions at temperatures near 50 °C. Unfolding eliminated the LEF-3 domains that are resistant to proteolysis and randomized the digestion pattern by trypsin. The structural transition was irreversible and was accompanied by the generation of high molecular weight (MW) complexes. The thermal treatment inhibited DNA-binding and unwinding activity of LEF-3 but markedly stimulated its annealing activity. We propose that the shift in LEF-3 activities resulted from the generation of the high MW protein complexes, that specifically stimulate the annealing of complementary DNA strands by providing multiple DNA-binding sites and bringing into close proximity the interacting strands. The unfolded LEF-3 was active in a strand exchange reaction suggesting that it could be involved in the production of recombination intermediates

Differentiating inhibition selectivity and binding affinity of isocitrate dehydrogenase 1 variant inhibitors

Isocitrate dehydrogenase (IDH) 1/2 gain-of-function variants catalyze the production of the oncometabolite 2-hydroxyglutarate and are validated targets for leukemia treatment. We report binding and inhibition studies on 13 IDH1/2 variant inhibitors, including clinical candidates and drugs, with wild-type (wt) IDH1 and its cancer-associated variant, IDH1 R132H. Interestingly, all the variant inhibitors bind wt IDH1 despite not, or only weakly, inhibiting it. Selective inhibition of the IDH1 R132H variant over wt IDH1 does not principally relate to the affinities of the inhibitors for the resting forms of the enzymes. Rather, the independent binding of Mg2+ and 2-oxoglutarate to the IDH1 variant makes the variant more susceptible to allosteric inhibition, compared to the tighter binding of the isocitrate–Mg2+ complex substrate to wt IDH1. The results highlight that binding affinity need not correlate with inhibition selectivity and have implications for interpretation of inhibitor screening results with IDH and related enzymes using turnover versus binding assays

Liquid Extraction Surface Analysis for Native Mass Spectrometry: Protein Complexes and Ligand Binding

AbstractNative liquid extraction surface analysis (LESA) mass spectrometry enables the direct sampling of protein complexes from a solid surface. We have previously demonstrated native LESA mass spectrometry of holomyoglobin (∼17kDa) from glass slides and tetrameric haemoglobin (∼64kDa) from dried blood spots and thin tissue sections. Here, we further explore the capabilities of this emerging technique by investigating a range of proteins which exist in various oligomeric states in vivo. Tetrameric avidin (∼64kDa), octameric (∼190kDa) and hexadecameric (∼380kDa) CS2 hydrolase, and tetradecameric GroEL (∼800kDa) were all detected by native LESA mass spectrometry. Moreover, trimeric AmtB, a membrane protein, could also be observed by native LESA mass spectrometry. The suitability of LESA mass spectrometry for probing protein-ligand binding was also investigated. Non-covalent complexes of the ligand biotin with the proteins avidin, haemoglobin and bovine serum albumin were detected. The results indicate that non-specific binding is minimal and that native LESA mass spectrometry is a promising tool for the investigation of biologically significant ligand binding

Selected topics in e^+e^- collisions

Content : 1) The leading twist pion wave function, 2) "Improved" QCD sum rules with non-local condensates, 3) Pion and kaon form factors and charmonium decays: theory vs experiment, 4) \gamma^{*}\gamma\pi^{o} - form factor, 5) The new non-local axial anomaly, 6) Cross sections \gamma\gamma --> \pi^+\pi^-, K^+K^-, K_S K_S .Comment: Talk given at the International Workshop "e^+e^- collisions from \phi to J/\psi", March 1, 2006, Novosibirsk, Russia; 20 pages, 7 figures; v2: sect.2 expanded to clarify the argumentation, a number of small improvements in the tex

Formation conditions and parameters of mini-landslides on agricultural slope landscapes

The paper investigated the stability of agricultural land slopes to mini-shear landslides on the basis of a numerical assessment of the influence of soil parameters in the Volga-Vyatka region of Russia on its frictional properties. It has been established that at a moisture content of 0.30 ± 0.04 m3/m3 of gray forest soil and 0.22 ± 0.04 m3/m3 of soddy-podzolic soil, the contribution of soil stickiness to the soil friction coefficient was the most significant, which was due both to the aggregation of soil particles and to the destruction of soil capillaries, and corresponded to the lower limit of soil plasticity. On the basis of the ratio proposed in the paper, a numerical assessment of the ratio of the power and length of the landslide along the slope for different slope angles was carried out, the results of which correspond to real landslide processes on natural and artificial slopes of agricultural land