How Ubiquitin Unfolds after Transfer into the Gas Phase

The structural evolution of ubiquitin after transfer into the gas phase was studied by electron capture dissociation. Site-specific fragment yields show that ubiquitin’s solution fold is overall unstable in the gas phase, but unfolding caused by loss of solvent is slowest in regions stabilized by salt bridges

Activated Ion Electron Capture Dissociation (AI ECD) of proteins: synchronization of infrared and electron irradiation with ion magnetron motion.

Here, we show that to perform activated ion electron capture dissociation (AI-ECD) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with a CO(2) laser, it is necessary to synchronize both infrared irradiation and electron capture dissociation with ion magnetron motion. This requirement is essential for instruments in which the infrared laser is angled off-axis, such as the Thermo Finnigan LTQ FT. Generally, the electron irradiation time required for proteins is much shorter (ms) than that required for peptides (tens of ms), and the modulation of ECD, AI ECD, and infrared multiphoton dissociation (IRMPD) with ion magnetron motion is more pronounced. We have optimized AI ECD for ubiquitin, cytochrome c, and myoglobin; however the results can be extended to other proteins. We demonstrate that pre-ECD and post-ECD activation are physically different and display different kinetics. We also demonstrate how, by use of appropriate AI ECD time sequences and normalization, the kinetics of protein gas-phase refolding can be deconvoluted from the diffusion of the ion cloud and measured on the time scale longer than the period of ion magnetron motion

Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

Lasso peptides constitute a class of bioactive peptides sharing a knotted structure where the C-terminal tail of the peptide is threaded through and trapped within an N-terminalmacrolactamring. The structural characterization of lasso structures and differentiation from their unthreaded topoisomers is not trivial and generally requires the use of complementary biochemical and spectroscopic methods. Here we investigated two antimicrobial peptides belonging to the class II lasso peptide family and their corresponding unthreaded topoisomers: microcin J25 (MccJ25), which is known to yield two-peptide product ions specific of the lasso structure under collisioninduced dissociation (CID), and capistruin, for which CID does not permit to unambiguously assign the lasso structure. The two pairs of topoisomers were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) upon CID, infrared multiple photon dissociation (IRMPD), and electron capture dissociation (ECD). CID and ECDspectra clearly permitted to differentiate MccJ25 from its non-lasso topoisomer MccJ25-Icm, while for capistruin, only ECD was informative and showed different extent of hydrogen migration (formation of c\bullet/z from c/z\bullet) for the threaded and unthreaded topoisomers. The ECD spectra of the triply-charged MccJ25 and MccJ25-lcm showed a series of radical b-type product ions {\eth}b0In{\TH}. We proposed that these ions are specific of cyclic-branched peptides and result from a dual c/z\bullet and y/b dissociation, in the ring and in the tail, respectively. This work shows the potentiality of ECD for structural characterization of peptide topoisomers, as well as the effect of conformation on hydrogen migration subsequent to electron capture

Quasifree Pion Electroproduction from Nuclei in the Δ\Delta Region

We present calculations of the reaction $A(e,e^\prime \pi N)B$ in the distorted wave impulse approximation. The reaction allows for the study of the production process in the nuclear medium without being obscured by the details of nuclear transition densities. First, a pion electroproduction operator suitable for nuclear calculations is obtained by extending the Blomqvist-Laget photoproduction operator to the virtual photon case. The operator is gauge invariant, unitary, reference frame independent, and describes the existing data reasonably well. Then it is applied in nuclei to predict nuclear cross sections under a variety of kinematic arrangements. Issues such as the effects of gauge-fixing, the interference of the $\Delta$ resonance with the background, sensitivities to the quadrupole component of the $\Delta$ excitation and to the electromagnetic form factors, the role of final-state interactions, are studied in detail. Methods on how to experimentally separate the various pieces in the coincidence cross section are suggested. Finally, the model is compared to a recent SLAC experiment.Comment: 27 pages in REVTEX, plus 22 PS figures embedded using psfig.sty (included), uuencode

AFLP markers for the R-gene in the flea beetle, Phyllotreta nemorum, conferring resistance to defenses in Barbarea vulgaris

A so-called R-gene renders the yellow-striped flea beetle Phyllotreta nemorum L. (Coleoptera: Chrysomelidae: Alticinae) resistant to the defenses of the yellow rocket Barbarea vulgaris R.Br. (Brassicacea) and enables it to use it as a host plant in Denmark. In this study, genetic markers for an autosomal R-gene, inherited as a single, dominant locus in flea beetles from the Danish locality "Kværkeby" are described, and a genetic linkage map around this particular R-gene is constructed, using the technique of AFLP (Amplified Fragment Length Polymorphism

A mini-twister variant and impact of residues/cations on the phosphodiester cleavage of this ribozyme class.

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a 13C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg2+ ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn2+ or Cd2+ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2′-OCH3-U5 modified twister ribozyme

Electron Capture Dissociation Mass Spectrometry of Tyrosine Nitrated Peptides

In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification.Here, we have investigated the electron capture dissociation (ECD) and collision-induced association (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains

Photo- and Electron-Production of Mesons on Nucleons and Nuclei

In these lectures I will show some results obtained with the chiral unitary approach applied to the photo and electroproduction of mesons. The results for photoproduction of $\eta \pi^0 p$ and $K^0 \pi^0 \Sigma^+$, together with related reactions will be shown, having with common denominator the excitation of the $\Delta(1700)$ resonance which is one of those dynamically generated in the chiral unitary approach. Then I will show results obtained for the $e^+ e^- \to \phi f_0(980)$ reaction which reproduce the bulk of the data except for a pronounced peak, giving support to a new mesonic resonance, X(2175). Results will also be shown for the electromagnetic form factors of the $N^*(1535)$ resonance, also dynamically generated in this approach. Finally, I will show some results on the photoproduction of the $\omega$ in nuclei, showing that present experimental results claiming a shift of the $\omega$ mass in the medium are tied to a particular choice of background and are not conclusive. One the other hand, the same experimental results show unambiguously a huge increase of the $\omega$ width in the nuclear medium.Comment: Lecture at the "International School of Nuclear Physics", 29th Course Quarks in Hadrons and Nuclei, Erice, Italy, September 2007. Note added in Proofs concerning the mixed events technique and other comments on omega productio

Electromagnetic Meson Production in the Nucleon Resonance Region

Recent experimental and theoretical advances in investigating electromagnetic meson production reactions in the nucleon resonance region are reviewed.Comment: 75 pages, 42 figure

Photo- and Electroproduction of Eta Mesons

Eta photo- and electroproduction off the nucleon is investigated in an effective lagrangian approach that contains Born terms and both vector meson and nucleon resonance contributions. In particular, we review and develop the formalism for coincidence experiments with polarization degrees of freedom. The different response functions appearing in single and double polarization experiments have been studied. We will present calculations for structure functions and kinematical conditions that are most sensitive to details of the lagrangian, in particular with regard to contributions of nucleon resonances beyond the dominant $S_{11}$(1535) resonance.Comment: 24 pages RevTeX/LaTeX2.09, NFSS1, 13 figures (in separate file (tar,gzip and uue)), accepted for publication in Z. Phys.