Tandem affinity purification (TAP) allows for rapid and efficient purification of epitope-tagged protein complexes from crude extracts under native conditions. The method was established in yeast and has been successfully applied to other organisms, including mammals and trypanosomes. However, we found that the original method, which is based on the TAP tag, consisting of a duplicate protein A epitope, a tobacco etch virus protease cleavage site, and the calmodulin-binding peptide (CBP), did not yield enough recovery of transcription factor SNAPc (for small nuclear RNA-activating protein complex) from crude trypanosome extracts for protein identification. Specifically, the calmodulin affinity chromatography step proved to be inefficient. To overcome this problem, we replaced CBP by the protein C epitope (ProtC) and termed this new epitope combination PTP tag. ProtC binds with high affinity to the monoclonal antibody HPC4, which has the unique property of requiring calcium for antigen recognition. Thus, analogous to the calcium-dependent CBPcalmodulin interaction, ProtC-tagged proteins can be released from immobilized HPC4 by a chelator of divalent cations. While this property was retained, epitope substitution improved purification in our experiments by eliminating the inefficiency of calmodulin affinity chromatography and by providing an alternative way of elution using the ProtC peptide in cases where EGTA inactivated protein function. Furthermore, HPC4 allowed highly sensitive and specific detection of ProtC-tagged proteins after protease cleavage. Thus far, we have successfully purified and characterized the U1 small nuclear ribonucleoprotein particle, the transcription factor complex TATA-binding protein related factor 4 (TRF4)/SNAPc/transcription factor IIA (TFIIA), and RNA polymerase I of Trypanosoma brucei.Tandem affinity purification (TAP) has been developed as an efficient tool for protein complex purification under nondenaturing conditions (23,24). For this method, the protein of interest must be fused to the TAP tag and expressed in the organism or cell line under investigation. The TAP tag contains two immunoglobulin G (IgG)-binding domains of the Staphylococcus aureus protein A (ProtA) and the calmodulinbinding peptide (CBP). Both epitope types are separated by spacer regions and a cleavage site for tobacco etch virus (TEV) protease. In consecutive steps, TAP is achieved by binding of the tagged protein to an IgG column, release of the protein by TEV protease cleavage, binding of the CBP-tagged protein to a calmodulin column, and final elution of the bound protein by a buffer containing a chelating agent of divalent cations such as EGTA. The final elution step is facilitated by calcium cation dependence of CBP binding to calmodulin. The TAP method has been developed in the budding yeast Saccharomyces cerevisiae, and it has been outstandingly successful in this organism, enabling a thorough proteome characterization by systematic analysis of protein complexes (8). TAP has two great advantages over c...

Insightful guide to developing efficient gas diffusion electrodes for electrochemical CO2 conversion to fuels and chemicals.

Luminescent lanthanide(III)-based molecular scaffolds hold great promises for materials science and for biological applications. Their fascinating photophysical properties enable spectral discrimination of emission bands that range from the visible to the near-infrared (NIR) regions. In addition, their strong resistance to photobleaching makes them suitable for long duration or repeated biological experiments using a broad range of sources of excitation including intense and focalized systems such as lasers (e.g., confocal microscopy). A main challenge in the creation of luminescent lanthanide(III) complexes lies in the design of a ligand framework that combines two main features: (i) it must include a chromophoric moiety that possesses a large molar absorptivity and is able to sensitize several different lanthanide(III) ions emitting in the visible and/or in the near-infrared, and (ii) it must protect the Ln(3+) cation by minimizing nonradiative deactivation pathways due to the presence of -OH, -NH and -CH vibrations. Herein, a new family of luminescent Ga(3+)/Ln(3+) metallacrown (MC) complexes is reported. The MCs with the general composition [LnGa4(shi)4(C6H5CO2)4(C5H5N) (CH3OH)] (Ln-1, Ln = Sm(3+)-Yb(3+)) were synthesized in a one pot reaction using salicylhydroxamic acid (H3shi) with Ga(3+) and Ln(3+) nitrates as reagents. The molecular structure of [DyGa4(shi)4(C6H5CO2)4(C5H5N) (CH3OH)] was obtained by X-ray analysis of single crystals and shows that the complex is formed as a [12-MCGa(III)shi-4] core with four benzoate molecules bridging the central Dy(3+) ion to the Ga(3+) ring metals. The powder X-ray diffraction analysis demonstrates that all other isolated complexes are isostructural. The extended analysis of the luminescence properties of these complexes, excited by the electronic states of the chromophoric ligands, showed the presence of characteristic, sharp f-f transitions that can be generated not only in the NIR (Sm, Dy, Ho, Er, Yb) but also in the visible (Sm, Eu, Tb, Dy, Tm). All Ln-1 complexes possess very high quantum yield values with respect to other literature compounds, indicating a good sensitization efficiency of the [12-MCGa(III)shi-4] scaffold. Especially, as of today, the Yb-1 complex exhibits the highest NIR quantum yield reported for a lanthanide(III) complex containing C-H bonds with a value of 5.88(2)% in the solid state. This work is a significant step forward toward versatile, easily prepared luminescent lanthanide(III) complexes suitable for a variety of applications including highly in demand biological imaging, especially in the NIR domain.

Electrochemical carbon dioxide (CO2) reduction powered by renewable electricity offers a path to produce valuable products from CO2 -this earth-scale human waste-and to store intermittent renewable energy in the form of chemical fuels. Recently, single metal atoms (SMAs) immobilized on a conductive substrate have been shown as effective catalysts for the electrochemical CO2 reduction, opening the door to a generation of low-cost and high-performance catalysts for fuel and chemical production. The unique physical and chemical properties of a single-atomic structure, the homogeneity of the active sites, combined with tunable coordination environments, are essential for realizing highly active and selective catalysts. In this review, we focus on the structure-performance relationship in SMA catalysts for CO2 reduction from both theoretical and experimental aspects. We discuss why SMA catalysts exhibit distinct catalytic performance compared to their counterpart nanoparticles. Recent strategies for improving the CO2 reduction selectivity and activity by tuning the nature and coordination environment of SMA active sites are described. Finally, we highlight potential applications of SMA catalysts in practical CO2 reduction 2 conditions, critical challenges, and the path toward efficient electrochemical CO2 reduction catalysis based on SMAs.

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