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Breakthrough of a Fluorogenic Probe for In Situ Pyruvate Kinase M2 Isoform (PKM2) Marking via Chemoselective SNAr with a Holding Internet site Amino acid lysine Residue.
Structural analysis of PiuA FeIII-bis-catechol and GaIII-bis-catechol and GaIII-(NE)2 complexes by NMR spectroscopy reveals only localized structural perturbations in PiuA upon ligand binding, largely consistent with recent descriptions of other solute binding proteins of type II ABC transporters. We speculate that tetradentate FeIII complexes formed by mono- and bis-catechol species are important Fe sources in Gram-positive human pathogens, since PiuA functions in the same way as SstD from Staphylococcus aureus.Rhodopsin is the photosensitive protein, which binds to 11-cis-retinal as its chromophore. In the dark, rhodopsin exists as a stable complex between the opsin moiety and 11-cis-retinal. The absorption of a light photon converts 11-cis-retinal to all-trans-retinal and initiates our vision. As a result, the increase in the rate of dark activation of rhodopsin reduces its photosensitivity resulting in night blindness. The mutations, G90D and T94I are night blindness-causing mutations that exhibit completely different physicochemical characteristics associated with the dark activation of rhodopsin, such as a high rate of thermal isomerization of 11-cis-retinal and a slow pigment regeneration. To elucidate the molecular mechanism by which G90D and T94I mutations affect rhodopsin dark activation and regeneration, we performed light-induced difference FTIR spectroscopy on dark and primary photo-intermediate states of G90D and T94I mutants. The FTIR spectra clearly show that both charged G90D and hydrophobic T94I mutants alter the H-bond network at the Schiff base region of the chromophore, which weakens the electrostatic interaction with Glu113 counterion. Our results further show an altered water-mediated H-bond network around the central transmembrane region of mutant rhodopsin, which is reminiscent of the active Meta-II state. This altered water-mediated H-bond network may cause thermal isomerization of the chromophore and facilitate rhodopsin dark activation.Estrogen-related receptor β (ERRβ) is a nuclear receptor critical for many biological processes. Despite the biological and pharmaceutical importance of ERRβ, deciphering the structure of ERRβ has been hampered by the difficulties in obtaining a pure and stable protein for structural studies. In fact, the ERRβ ligand-binding domain remains the last unsolved ERR structure and also one of only a few unknown nuclear receptor structures. Here, we report the identification of a critical single-residue mutation resulted in robust solubility and stability of an active ERRβ ligand-binding domain, thereby providing a protein tool enabling the first probe into the biochemical and structural studies of this important receptor. find more The crystal structure reveals key structural features that have enabled the integration of the molecular determinants of signals transduced across the ligand binding and coregulator recruitment by all three ERR subtypes, which also provides a framework for the rational design of selective and potent ligands for the treatment of various ERR-mediated diseases.One-carbon metabolism produces methionine and N10-formyl-tetrahydrofolate (N10-fTHF) required for aminoacylation and formylation of initiator tRNA (i-tRNA), respectively. In Escherichia coli, N10-fTHF is made from 5, 10-methylene-THF by a two-step reaction using 5,10-methylene-THF dehydrogenase/cyclohydrolase (FolD). The i-tRNAs from all domains of life possess a highly conserved sequence of three consecutive G-C base pairs (3GC pairs) in their anticodon stem. A 3GC mutant i-tRNA (wherein the 3GC pairs are mutated to those found in elongator tRNAMet) is incompetent in initiation in E. coli (even though it is efficiently aminoacylated and formylated). Here, we show that E. coli strains having mutations in FolD (G122D or C58Y or P140L) allow a plasmid encoded 3GC mutant i-tRNA to participate in initiation. In vitro, the FolD mutants are highly compromised in their dehydrogenase/cyclohydrolase activities leading to reduced production of N10-fTHF and decreased rates of i-tRNA formylation. The perturbation of one-carbon metabolism by trimethoprim (inhibitor of dihydrofolate reductase) phenocopies FolD deficiency and allows initiation with the 3GC mutant i-tRNA. This study reveals an important crosstalk between one-carbon metabolism and the fidelity of translation initiation via formylation of i-tRNA, and suggests that augmentation of the age old sulfa drugs with FolD inhibitors could be an important antibacterial strategy.Transcription factors are often the downstream effectors of signaling cascades. In fission yeast, the transcription factor Atf1 is phosphorylated by the MAP kinase Sty1 under several environmental stressors to promote transcription initiation of stress genes. However, Sty1 and Atf1 have also been involved in other cellular processes such as homologous recombination at hotspots, ste11 gene expression during mating and meiosis, or regulation of fbp1 gene transcription under glucose starvation conditions. Using different phospho-mutants of Atf1, we have investigated the role of Atf1 phosphorylation by Sty1 in those biological processes. An Atf1 mutant lacking the canonical MAP kinase phosphorylation sites cannot activate fbp1 transcription when glucose is depleted, but it is still able to induce recombination at ade6.M26 and to induce ste11 after nitrogen depletion; in these last cases, Sty1 is still required, suggesting that additional non-canonical sites are activating the transcription factor. In all cases, an Atf1 phosphomimetic mutant bypasses the requirement of the Sty1 kinase in these diverse biological processes, highlighting the essential role of the DNA binding factor Atf1 on chromatin remodeling and cell adaptation to nutritional changes. We propose that post-translational modifications of Atf1 by Sty1, either at canonical or non-canonical sites, are sufficient to activate some of the functions of Atf1, those involving chromatin remodeling and transcription initiation. However, in the case of fbp1 where Atf1 acts synergistically with other transcription factors, elimination of the canonical sites is sufficient to hamper some of the interactions required in this complex scenario and to impair transcription initiation.
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