Ligand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry

Background Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. Methods/Findings In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Conclusions/Significance Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.METASU

Revealing Higher Order Protein Structure Using Mass Spectrometry

International audienceThe development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope

Dispositifs originaux d'un monochromateur infrarouge (1-33 μ) pour études multiples

We have built a high performance infrared monochromator allowing to measure the 1 μ-33 μ range spectra. This classical instrument presents some new optical and mechanical devices. We have used it to study the anharmonicity of ionic and molecular compounds, and the structures of some crystals.Nous avons construit un monochromateur infrarouge à performances élevées permettant d'explorer une zone du spectre s'étendant de 1 μ à 33 μ. Cet appareil, de conception classique, comporte des dispositifs optiques et mécaniques originaux. Il a été utilisé avec succès dans le cas des études des propriétés anharmoniques des cristaux ioniques et moléculaires ainsi que pour préciser les structures de certains cristaux

Polyacetylene thin film photovoltaic devices

Photovoltaic devices have been obtained by a direct polymerization of undoped (or p-type doped) thin film (CH)x layer onto a polycrystalline cadmium sulfide film. The morphology of the contact is observed by S.E.M. microscopy. The electrical characteristics of this device are studied in the dark or under illumination. The main result, at AM1, 100 mW/cm2 with doped (CH)x correspond to 0.5 percent efficiency.Des dispositifs photovoltaïques ont été fabriqués par polymérisation directe de films minces de polyacétylène dopé ou non dopé sur une lame mince polycristalline de CdS déposée sur verre conducteur. La morphologie du contact est déterminée par microscopie à balayage. Les caractéristiques électriques ont été obtenues à l'obscurité et sous éclairement. Dans des conditions standard (AM1, 100 mW/cm2), les hétérojonctions entièrement couches minces à base de (CH) x dopé atteignent un rendement de 0,5 %