National Institute For Advanced Industrial Science & Technology, Aist, Tsukuba, Japan Work Values

Daily Duties at National Institute For Advanced Industrial Science & Technology, Aist, Tsukuba, Japan:

1. HutP protein from Bacillus subtilis were purified to homogeneity using ACTA purifier and dialyzed against the binding buffer (15mM Hepes, pH 7.4 containing 150mM NaCl and 10mM MgCl2) and used for further studies. The full-length intervening sequence (+489 – +600 between the stop codon of hutP and the initiation codon of the hutH gene) contains hut terminator region. In order to determine whether the HutP recognizes two potential binding sites within the RNA for destabilizing the terminator structure of hut operon, various kinds of RNAs (55-mer) having two potential binding sites were prepared and complex crystals were obtained by hanging drop vapor diffusion method which diffracted beyond 2.0 Å resolution. The Hutp complex crystals are monoclinic with space group C2 and cell dimensions a = 100.306Å, b= 76.847Å, c = 63.066Å and β = 109.08. The structure solution was obtained by molecular replacement method using Hutp complex with 21-mer rna (PDB id: 1WMQ) as starting model, using the program AMoRe implemented in CCP4 suite (Collaborative Computational Project, No. 4, 1994). Structure refinement was done using REFMAC5.0 and modeled using XtalView program. The final refined model consists of one trimer (chains A, B and C) and 21-mer rna. The final refined model has an R factor of 23.6% (Rfree = 29.5%). The final electron density is of excellent quality, wherein the continuous electron density for the 21-mer RNA was observed on the surface of the trimer. The Hutp complex structure has been submitted to the protein data bank. 2. B.cereus Hutp protein was purified, crystallized and the data collection was done. The crystal diffracted to around 3.5Å resolution, since the resolution is very low the quality of the crystal is being improved by point mutation. 3. L-lactate oxidase (LOX) catalyzes the flavin mononucleotide-dependent oxidation of lactate to pyruvate and hydrogen peroxide. The abstraction of a proton from the substrate is widely accepted as the initial step of the reaction; however, the details of the reaction mechanism have remained elusive. We have revealed the reaction mechanism, based on the crystal structure of LOX complexed with its substrate analogue at 1.59Å resolution. To confirm the enzymatic reaction mechanism, four different mutants were designed and mutants of LOX were constructed at positions 174 and 221 (D174A, D174N, K221A, and K221S), by PCR-based site-directed mutagenesis, using a Quick Change Site-Directed Mutagenesis Kit (Stratagene). The K221S mutant was tested, because the serine residue is highly conserved among the other flavoenzymes. The wild-type and mutants of LOX were expressed in E.coli JM109, and were purified to homogeneity using ACTA purifier (Amersham Biosciences). The results revealed that the four mutants completely lost the enzymatic activity thus supporting the reaction mechanism. 4. The crystal structure of a self-complementary RNA duplex r(GGCGBrUGCGCU)2 with terminal G·U and internal tandem G·U base pairs has been determined at 2.1 Å resolution.

What they like about National Institute For Advanced Industrial Science & Technology, Aist, Tsukuba, Japan:

Working in an organization that is technologically advanced, creative, and innovative is critical in your assessment of hiring companies. Less important to you are stability of the organization, the length of time an organization has been in business, and the business' plans for the future. You prefer a fast-moving company that will take risks to achieve its goals and objectives. The length of time the company has been in business is irrelevant. Maintaining status quo is unnecessary. You thrive on change, uncertainty and the upside of potential business risks, especially those associated with innovation. Stability for the long haul is not nearly as important as is working in an atmosphere that is charged with a sense of urgency and constant change.

Tags

biochemistry, structural biology, scientist, membrane protein, crystallography, protein purification, protein crystallization, synchrotron data collection, molecular biology

Skills

membrame protein crystallography, Protein purification using HPLC, AKTA purifier, protein crystallization, Genomic DNA purification, Molecular Cloning, Protein Expression, purification and characterization of recombinant proteins, including selenomethionine-substituted proteins. , Membrane protein expression, purification and crystallization., Characterization of proteins using electrophoresis (native, non-reduced and Western blotting) and spectroscopic (UV/VIS and fluorescence) techniques., DNA-protein, RNA-protein crystallization., X-ray diffraction experiment and data collection (PF-KEK, Tsukuba, Japan & APS - Chicago, USA ). , Expertise in data collection techniques using mar345 image plate detectors and data reduction software such as DENZO, Scalepack, HKL2000, MOSFLM and AUTOMAR., Protein Phasing by MR and SAD/MAD technique using crystallographic programs, SHARP, CCP4, SHELXD and Solve/Resolve., Proficient in using structure refinement and model building software namely CNS, CCP4, PHENIX, SHELXE, ARP/wARP and analyze the structure using software O, CCP4mg and Coot., Proficient in small molecule structure analysis packages (SHELXS, SHELXL, ORTEP and PLATON)., Expertise in molecular modeling and graphics software such as Insight II- Biosym software (Silicon Graphics), Pymol, Swiss-PDB viewer, RasMol, Molscript, Bobscript, O, Coot, XtalView etc., Experience in using Insight II Biosym Softwares for drug docking and Molecular Mechanics studies and Homology modeling. , Expertise in operating system Windows, Linux and Unix, MALS ( Multiple angle dynamic light scattering), electrophysiology - patch clamp

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