Mogens Brøndsted Nielsen

A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.

Mass spectrometry–based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.

Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases

We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling molecule in the epidermal growth factor receptor signaling pathway. This molecule, designated Odin, contains several ankyrin repeats, two sterile alpha motifs and a phosphotyrosine binding domain and is ubiquitously expressed. Using antibodies against endogenous Odin, we show that it undergoes tyrosine phosphorylation upon addition of growth factors such as EGF or PDGF but not by cytokines such as IL-3 or erythropoietin. Immunofluorescence experiments as well as Western blot analysis on subcellular fractions demonstrated that Odin is localized to the cytoplasm both before and after growth factor treatment. Deletion analysis showed that the phosphotyrosine binding domain of Odin is not required for its tyrosine phosphorylation. Overexpression of Odin, but not an unrelated adapter protein, Grb2, inhibited EGF-induced activation of c-Fos promoter. Microinjection of wild-type or a mutant version lacking the PTB domain into NIH3T3 fibroblasts inhibited PDGF-induced mitogenesis. Taken together, our results indicate that Odin may play a negative role in growth factor receptor signaling pathways.

An enlarged bis-bipyridinium cyclophane-Au nanoparticle superstructure for selective electrochemical sensing applications

Highly stable electrostatically-linked superstructures of 13 nm Au-colloids have been constructed by the use of the tetracationic cyclophanes cyclobis(paraquat-p-phenylene) and cyclobis(paraquat-p-biphenylene). These architectures have been characterized by optical and electrochemical means and exhibit sensing capabilities that are shown to depend on the crosslinking cyclophane. While superstructures linked by cyclobis(paraquat-p-phenylene) sense hydroquinone derivatives and not ferrocene derivatives, those linked by the larger receptor cyclobis(paraquat-p-biphenylene) sense only the ferrocene derivatives. This high degree of selectivity is based on the topological and supramolecular fit of the analyte in the cavity of the receptor. Arrays containing both cyclophanes are shown to exhibit sensing characteristics that are dependent on the position of each cyclophane in the superstructure, possibly as a consequence of the limited porosity of the composite.

Synthesis and non-linear optical properties of mono-pyrrolotetrathiafulvalene derived donor–π–acceptor dyads

The synthesis of novel donor–π–acceptor dyads based on a pyrrolo-annelated tetrathiafulvalene unit is described. The linear and non-linear optical properties of these chromophores have been investigated, together with their redox properties. For a dyad containing a p-nitrophenyl acceptor group, a significant third-order optical non-linearity is observed.

Self-Complexing Tetrathiafulvalene-Based Donor–Acceptor Macrocycles

Two “self-complexing” macrocycles (22ab and 23ab) based on a methylthio-substituted derivative of the electron donor tetrathiafulvalene (TTF) and a cyclic bipyridinium acceptor have been prepared.[1] When “decomplexed” by fractional crystallization, the rigid compound 22b was not able to undergo “recomplexation” to any significant degree, whereas for the more flexible 23b, an equilibrium between “complexed” (23a) and “uncomplexed” (23b) compounds was slowly re-established in solution according to UV/Vis measurements. 23ab was thus seen to behave as a “thermo-switch”, although with a rather slow response. Any complexation between the two separated components, i.e. the cyclic acceptor 3 and the tetrakis(methylthio)tetrathiafulvalene 6, was not observed by 1H-NMR spectroscopy. However, 3 was able to bind unsubstituted TTF (5) in its cavity, albeit with a small association constant of only 60 M–1. Taking advantage of the tetravalency of TTF, we also report the synthesis of a “self-complexing” pyromellitic diimide/TTF macrocycle (33ab). Whereas 22ab and 23ab were prepared employing the concept of template-assisted synthesis,[2] the synthesis of 33ab did not rely on this technique.

The tetrathiafulvalene dication in the gas phase: its formation and stability

The outcome of high-energy collisions of tetrathiafulvalene radical cations, TTF˙+, strongly depends on the nature of the collision gas. Thus, bond cleavage of the central fulvalene bond is promoted by an inert gas, such as helium, whereas oxidation to the dication is a significant reaction channel when molecular oxygen is the collision gas. DFT calculations (B3LYP/6-311++G(2d,p)//B3LYP/6-31+G(d)) indicate that TTF2+ is both thermodynamically and kinetically stable with respect to dissociation into two singly charged dithiole ring fragments or a doubly charged and a neutral dithiole.

Tetrathiafulvalene-containing pseudorotaxanes formed between dibenzylammonium salts and crown ethers

Abstract The synthesis of two tetrathiafulvalene (TTF) derivatives, one carrying arms incorporating four dibenzylammonium centers and the other encompassing two annulated crown ether rings—i.e., hydrogen bond donor and acceptor functions, respectively, is described. In the presence of macrocyclic polyethers—e.g., DB24C8 and BPP34C10—and dibenzylammonium hexafluorophosphate, these two TTF derivatives form pseudorotaxanes as evidenced by 1H NMR spectroscopy, mass spectrometry, and differential pulse voltammetry.

Synthesis of linear oligo-TTFs and their [2]rotaxanes

Two linear oligo-TTFs were synthesised employing a stepwise strategy involving two different thiolate protecting groups. These linear TTFs were incorporated into donor–acceptor rotaxanes with the cyclic acceptor, cyclobis(paraquat-p-phenylene). Moreover, a prototype rotaxane based on a bis(pyrrolo)-TTF was prepared and studied.

Ultra-high pressure synthesis of a tetrathiafulvalene-diquat cyclophane

Abstract Quaternization of a tetrathiafulvalene (TTF)/2,2′-bipyridine cyclophane under 10 kbar of pressure yields a TTF–diquat donor–acceptor cyclophane revealing distinct charge-transfer interactions.

Experimental evidence for the 7,7,8,8-tetracyano-p-quinodimethane dianion in vacuo

The existence of the unsolvated 7,7,8,8-tetracyano-p-quinodimethane (TCNQ) dianion is proved experimentally. The dianion was formed in 50-keV collisions between TCNQ monoanions and sodium vapor. In the collision process, electron capture to the monoanion occurs with a cross section of about 1 A2. The lifetime of the dianion is estimated to be in the order of microseconds or longer as the flight time from the collision cell to the detector is 5 μs. The stability of the dianion was elucidated by density functional theory (DFT) and ab initio MP2 calculations. A selection of dissociation reactions have been studied theoretically and compared with the experimental results.