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  • [Crystallization] X-ray crystallographic studies of a fluorescent protein Akane1 from Scleronephthya gracillimum

    Fluorescent proteins (FPs) are popular tools used to monitor the localization of a biomolecule and its interaction (in vivo or in vitro). Changes in fluorescence intensity caused by environmental elements (e.g. protons or metals) form the basis for the development of pH indicators or metal biosensors. FPs are constantly being engineered or discovered for efficient and extended applications. In this study, we report the expression, purification, characterization, crystallization, and preliminary X-ray diffraction studies of a fluorescent protein Akane1 from Scleronephthya gracillimum. This protein exhibited a dimeric state in solution. According to the spectral analysis, Akane1 showed excitation and emission maxima peaks at 501 and 510 nm, respectively. The crystals of Akane1 were obtained under a reservoir solution containing 100 mM Tris-HCl (pH 7.0), 200 mM calcium acetate, and 20% (w/v) PEG 3000. The crystals of Akane1 diffracted to 1.93 Å and belonged to the space group P21, with unit cell parameters of a = 56.02 Å, b = 126.67 Å, c = 62.03 Å, and β = 107.52°. The initial phase of Akane1 was obtained by the molecular replacement method.

  • [Crystallization] Crystallization and preliminary diffraction analysis of human TEAD1, a transcriptional enhancer factor that controls the Hippo signaling pathway

    Transcriptional enhancer activation domain (TEAD) proteins are transcription factors that promote the expression of genes involved in organogenesis, embryonic development, and tumorigenesis. TEAD functions downstream of the Hippo signaling pathway as one of the pivotal regulators that controls cell viability and proliferation, tissue growth, and organ size, by interacting with yes-associated protein (YAP) via its C-terminal YAP-binding domain (YBD). As YAP is a wellknown oncoprotein and its interaction with TEAD has been shown to be critical for the function of YAP, TEAD proteins are emerging as a potential therapeutic target for cancer. In this study, the YBD of the TEAD1 protein was produced from an Escherichia coli expression system, purified using a Ni-NTA affinity chromatography, HiTrap Q anion exchange chromatography, and size exclusion chromatography, and then successfully crystallized. X-ray diffraction data were collected to the resolution of 1.70 Å. Preliminary diffraction analysis revealed that the TEAD1 YBD crystals belong to the space group P212121 with unit cell parameters of a = 36.5 Å, b = 89.4 Å, c = 135.6 Å, and that two TEAD1 YBD molecules are contained in the asymmetric unit with a solvent content of 45.2%.

  • [Crystallization] Aeromonas hydrophila cytosolic 5’-methylthioadenosine/S-adenosylhomocysteine nucleosidase MtaN-2: crystallization and X-ray crystallographic analysis

    5’-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) plays a critical role in diverse pathways in bacterial cells such as biological methylation, polyamine biosynthesis, methionine recycling, and bacterial quorum sensing. It has been known that MtaN catalyzes the hydrolysis of N-ribosidic bond of adenosine-based substrates such as S-adenosyl-Lhomocysteine (SAH), S-methyl-5’-thioadenosine (MTA) and 5’-deoxyadenosine (5’-DOA). In Aeromonas hydrophila, there are two MtnN subfamily proteins: MtaN-1, a periplasmic protein with an N-terminal signal peptide; and MtaN-2, a cytosolic protein. In this study, MtaN-2 from A. hydrophila was successfully expressed and purified using Ni-NTA affinity, Q anionexchange, and gel-filtration chromatography. We first crystallized apo MtaN-2 but it diffracted to a low resolution of 5.1 Å. New crystals suitable for diffraction were obtained by adding 2 mM adenosine, a substrate analog of MtaN-2 during purification process and the crystals diffracted to the resolution of 2.0 Å. The crystals belong to the trigonal space group P31 or P32, with unit-cell parameters of a = b = 74.94 Å and c = 185.21 Å. The asymmetric unit contains four complexes of MtaN-2 with hydrolysis products of adenosine.

  • [Crystallization] Phase determination of the UDP-Nacetylmuramic acid:L-alanine ligase (MurC) crystal from Mycobacterium bovis

    Bacterial peptidoglycan is necessary for bacterial survival against environmental osmotic pressure and is a relevant target for development of anti-bacterial drugs. Formation of a covalent bond between a carbohydrate and an amino acid is a key chemical process for peptidoglycan bio-synthesis. UDP-N-acetylmuramic acid (UDP-MurNAc):L-alanine ligase (MurC) is an ATP-dependent amide bond ligase to form an UDP-MurNAc-L-alanine required in bacterial peptidoglycan. To provide a structural background for development of tuberculosis-specific antibiotics, Mycobacterium bovis MurC (MbMurC), which has sequence identity of 100% to M. tuberculosis, was cloned and expressed. The purified protein was crystallized from the precipitant of 0.1 M HEPES (pH 7.0), 0.2 M NaCl, 24% (w/v) polyethylene glycol 1.5K, and 10% (v/v) 2-methyl-2,4- pentanediol. Diffraction data were collected to 2.3 Å resolution. The crystal belonged to the primitive monoclinic space P21 with unit-cell parameters a = 65.30 Å, b = 76.70 Å, c =103.96 Å, α = γ = 90°, and β = 106.0°. The spatial positions of the two protein molecules in the asymmetric unit were determined by molecular replacement using the sequentially related Yersinia pestis MurC structure.

  • [Crystallization] Purification, crystallization, and X-ray crystallographic analysis of histidine triad nucleotide-binding protein from Candida albicans

    Histidine triad nucleotide-binding protein (HINT) is a member of the histidine triad (HIT) superfamily, and exhibits dinucleotide hydrolase activity via a histidine triad motif. HITs are divided into five classes that have various functions including galactose metabolism, DNA repair, and tumor suppression. Although multiple crystal structures have been reported, limited structural information is available for HINT proteins of fungal species. In this study, to understand the structural features and functional diversity of HINT proteins, we crystallized HINT from four fungal species and obtained X-ray diffraction data from the pathogenic fungus Candida albicans at a resolution of 2.5 Å. The crystal of the C. albicans HINT (CaHINT) belonged to the space group of P2221, with unit cell parameters a = 40.4, b = 101.9, c = 175.2 Å, α = β = γ = 90°. The crystal contained four macromolecules in asymmetric units.

  • [Crystallization] Expression, purification and crystallization of GSK3β in complex with the flavonoid morin

    GSK3β is an important kinase that functions in cellular signaling pathways. Morin, a flavonoid that is plentiful in nature, was found as an inhibitor of GSK3β that can reduce tau pathology in vivo and in vitro. To identify how morin inhibits GSK3β, GSK3β protein was overexpressed and purified using affinity and ion exchange chromatography. GSK3β protein was crystallized with morin using hanging drop vapor diffusion method in the presence of 18 % (v/v) PEG 4000, 100 mM sodium citrate (pH 6.5), and 5 % (v/v) 2-propanol at 290 K. X-ray diffraction data was collected to a maximum resolution of 2.14 Å. The crystal belonged to P21, with unit cell parameters a = 67.6 Å, b = 134.4 Å, c = 100.4 Å, α = γ = 90.0°, β = 103.8°.