Intrinsic Fluorometric Reporters of Pteridine Reductase 1, a Target for Antiparasitic Agents

The intrinsic steady-state and time-resolved fluorescence of Leishmania major pteridine reductase 1, a tetrameric protein target for anti-infective agents, is investigated and deciphered in terms of the contributions from populations of the two tryptophans included in each protein monomer. Signals from these local fluorometric reporters contain molecular-level information on the conformational landscape of this protein and on its interaction with a nanomolar pteridinic inhibito

Large third-order nonlinear optical response of porphyrin J-aggregates oriented in self-assembled thin films

The preparation and characterization of a self-assembled material showing a high nonlinear response and good photostability to ultrashort laser pulses is presented. The material is built by alternate deposition of tetrakis(4-sulfonatophenyl) porphyrin diacid (H4TPPS2-) and poly(diallyldimethylammonium chloride) (PDDA) forming electrostatically self-assembled multilayers (ESAMs). UV-visible absorption and emission experiments show that in this matrix H4TPPS2- is present mainly in its J-aggregated form. Furthermore, linear dichroism experiments on a 3 bilayer film show a preferential alignment of the porphyrin aggregate with the J-band transition dipole moment parallel to the film surface. The two photon absorption (TPA) properties of these films are investigated with the Z-scan technique at 806 nm, employing 130 fs pulses. The samples exhibit strong nonlinearities with a very large two-photon absorption coefficient beta(TPA) of 50 cm GW(-1). The origin of this large response is investigated. It has been already demonstrated that aggregation enhances the molecular TPA cross section of H4TPPS2- from 30 to 1000 GM in water solution thanks to cooperative effects. In a 20 bilayer film a further increase by a factor of 1.7 is observed and explained in terms of preferential alignment of J-aggregates in the multilayer

Spectroscopic study of chromophores in ancient HIMT glass

We have studied a series of Late Roman glass fragments belonging to the peculiar group called HIMT (High Iron Magnesium Titanium glass), characterized by different color nuances, and previously chemically characterized [1-3]. XANES, UV-VIS and luminescence spectroscopies [4-6] are exploited for determining the distribution of the oxidation states of the two chromophores Fe and Mn in the ancient finds and in two synthetic glass specifically prepared with selected chemically compositions. Fe and Mn K-edge XANES spectra were collected in fluorescence mode at the GILDA-CRG beamline (ESRF, Grenoble, France). A dynamically and sagittally focussing monochromator with Si (311) crystals was used. Energy calibrations were achieved using Fe and Mn foils as references and the position of the first inflection point was taken at 7112.0 and 6538.0 eV respectively. The pre-edge region was extracted from the normalized spectra and analysed by least-square fitting of pseudo-Voight functions of equal width to the pre-edge spectral envelope. For each sample, the pre-edge centroid was calculated from the average position of the pseudo-Voigt functions, weighted by their respective integrated areas. UV-VIS absorption spectra were measured on a Varian Cary 100 double-beam spectrophotometer operated between 900 and 200 nm. Luminescence emission and excitation spectra were collected in a Spex Jobin-Yvon FluoroMax3 spectrofluorometer. On the basis of both XANES and UV-VIS results, in all the ancient glass samples Fe results to be mostly in its oxidate state 3+. XANES data indicate that the prevalent Mn oxidation state is 2+, however the presence of minor amounts of Mn3+ has been proved by UV-VIS and luminescence spectroscopy. The different spectroscopic techniques, used in a combined approach, are able to interpret the apparent anomalous colour of some of the ancient glass samples

Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology

Demonstrating a candidate drug's interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding