A general NMR method for rapid, efficient, and reliable biochemical screening

High-throughput screening is usually the method of drug-lead discovery. It is now well accepted that, for a functional assay, quality is more important than quantity. The ligand-based or protein-based NMR screening methodologies for detecting compounds binding to the macromolecular target of interest are now well established. A novel and sensitive NMR method for rapid, efficient, and reliable biochemical screening is presented. The method named 3-FABS (three fluorine atoms for biochemical screening) requires the labeling of the substrate with a CF(3) moiety and utilizes (19)F NMR spectroscopy for the detection of the starting and enzymatically modified substrates. The method allows for high-quality screening of large compound or natural product extract collections and for measuring their IC(50) values. Applications of this technique to the screening of inhibitors of the Ser/Thr kinase AKT1 and the protease trypsin are presented. In addition, an interesting application of 3-FABS to functional genomics is also presented.     

Sensitivity improvement in 19F NMR-based screening experiments: theoretical considerations and experimental applications

NMR-based binding and functional screening performed with FAXS (fluorine chemical shift anisotropy and exchange for screening) and 3-FABS (three fluorine atoms for biochemical screening) represents a potential alternative approach to high-throughput screening for the identification of novel potential drug candidates. The major limitation of this method in its current status is its intrinsic low sensitivity that limits the number of tested compounds. One approach for overcoming this problem is the use of a cryogenically cooled (19)F probe that reduces the thermal noise in the receiver circuitry. Sensitivity improvement in the two screening techniques achieved with the novel cryogenic (19)F probe technology permits an increased throughput, detection of weaker binders and inhibitors (relevant in a fragment-based lead discovery program), detection of slow binders, and reduction in protein and substrate consumption. These aspects are analyzed with theoretical simulations and experimental quantitative performance evaluation. Application of 3-FABS combined with the cryogenic (19)F probe technology to rapid screening at very low enzyme concentrations and the current detection limits reached with this approach are also presented.     

Synergic approach to XAFS analysis for the identification of most probable binding motifs for mononuclear zinc sites in metalloproteins

In the present work a data analysis approach, based on XAFS data, is proposed for the identification of most probable binding motifs of unknown mononuclear zinc sites in metalloproteins. This approach combines multiple‐scattering EXAFS analysis performed within the rigid‐body refinement scheme, non‐muffin‐tin ab initio XANES simulations, average structural information on amino acids and metal binding clusters provided by the Protein Data Bank, and Debye–Waller factor calculations based on density functional theory. The efficiency of the method is tested by using three reference zinc proteins for which the local structure around the metal is already known from protein crystallography. To show the applicability of the present analysis to structures not deposited in the Protein Data Bank, the XAFS spectra of six mononuclear zinc binding sites present in diverse membrane proteins, for which we have previously proposed the coordinating amino acids by applying a similar approach, is also reported. By comparing the Zn K‐edge XAFS features exhibited by these proteins with those pertaining to the reference structures, key spectral characteristics, related to specific binding motifs, are observed. These case studies exemplify the combined data analysis proposed and further support its validity.     

Time-dependent heat capacity of water-lysozyme solution

Summary We report here the first observation of the time-dependent heat capacity of water-lysozyme solution. The observed increase of heat capacity in time occurs independently of the lysozyme concentration and thepH value of the solvent. The time necessary to attain the heat capacity equilibrium value and the amplitude of the effect are not well reproducible; the former tends to increase with temperature, while the amplitude decreases and practically disappears about 35°C. Evolution of a spatial order of the proteins in water and/or of water molecules interacting with the proteins is considered as a possible explanation for the results. The phenomenon promises to be an important tool to study the role of water in biological organization and activity.     

The Legnaro Francium Magneto-Optical Trap

Hyperfine Interactions - Laser cooling and trapping of radioactive atoms represent the new frontier in atomic physics and a new powerful tool in nuclear physics. We are setting up at the...     

Spectroscopic properties and upconversion in Pr3+:YF3 nanoparticles

The synthesis and spectroscopic investigation of Pr(3+):YF(3) nanoparticles with nominal concentration between 0.05% and 5 at% Pr(3+) are reported. Pr(3+) emission in the visible range of the spectrum is investigated at room temperature and at 10 K as well as time resolved spectroscopy as a function of Pr(3+) concentration. The upconverted emission from the orange to the blue region is observed and the time-resolved spectroscopy of the visible emissions is discussed as a function of the doping level. A careful analysis of the decays permits identification of the main energy-transfer mechanisms that determine the population of the excited levels at various times during the decay.     

Fluorine-NMR competition binding experiments for high-throughput screening of large compound mixtures

High-throughput ligand-based NMR screening with competition binding experiments is extended to (19)F detection. Fluorine is a favorable nucleus for these experiments because of the significant contribution of the Chemical Shift Anisotropy (CSA) to the (19)F transverse relaxation of the ligand signal when bound to a macromolecular target. A low to moderate affinity ligand containing a fluorine atom is used as a reference molecule for the detection and characterization of new ligands. Titration NMR experiments with the selected reference compound are performed for finding the optimal set-up conditions for HTS and for deriving the binding constants of the identified NMR hits. Rapid HTS of large chemical mixtures and plant or fungi extracts against the receptor of interest is possible due to the high sensitivity of the (19)F nucleus and the absence of overlap with the signals of the mixtures to be screened. Finally, a novel approach for HTS using a reference molecule in combination with a control molecule is presented.     

Passively Q-switched Tm:YLF laser

Stable passive Q-switching of a Tm: LiYF4 laser is obtained using polycrystalline Cr2+: ZnS as a saturable absorber. The achieved maximum pulse energy of 0.9 mJ and peak power of 65 kW for a pulse duration of ～14 ns represent substantial improvement and highest values for a passively Q-switched diode-pumped Tm laser operating at ～1.9 μm.     

Impact of the analytical blank in the uncertainty evaluation of the copper content in waters by flame atomic absorption spectrometry

Chemical analysts use analytical blanks in their analyses, but seldom is this source of uncertainty evaluated. Generally, there is great confusion. Although the numerical value of the blank, in some situations, can be negligible, its source of uncertainty cannot be. This article discusses the uncertainty contribution of the analytical blank using a numerical example of the copper content in waters by flame atomic absorption spectrometry. The results indicate that the uncertainties of the analytical blank can contribute up to 50% when the blank sample is considered in this analysis, confirming its high impact. This effect can be primarily observed where the analyte concentration approaches the lower range of the analytical curve. Even so, the blank is not always computed. Therefore, the relevance of the analytical blank can be confirmed by uncertainty evaluation.