• Molecules and Cells
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• v.23 no.1
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• pp.100-107
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• 2007

The moss Physcomitrella patens has two life cycles, filamentous protonema and leafy gametophore. A modified from of suppression subtractive hybridization (SSH), mirror orientation selection (MOS), was applied to screen genes differentially expressed in the P. patens protonema. Using reverse Northern blot analysis, differentially expressed clones were identified. The identified genes were involved mainly in metal binding and detoxification. One of these genes was an AP2 (APETALA2) domain-containing protein (PpACP1), which was highly up-regulated in the protonema. Alignment with other AP2/EREBPs (Ethylene Responsive Element Binding Proteins) revealed significant sequence homology of the deduced amino acid sequence in the AP2/EREBP DNA binding domain. Northern analysis under various stress conditions showed that PpACP1 was induced by ethephon, cadmium, copper, ABA, IAA, and cold. In addition, it was highly expressed in suspension-cultured protonema. We suggest that PpACP1 is involved in responses to metals, and that suspension culture enhance the expression of genes responding to metals.

• Biomedical Science Letters
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• v.16 no.3
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• Bulletin of the Korean Chemical Society
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• v.29 no.4
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• pp.736-740
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• 2008

The interaction of myelin basic protein (MBP) from bovine central nervous system with divalent calcium ion was studied by isothermal titration calorimetry at 27 ${^{\circ}C}$ in aqueous solution. The extended solvation model was used to reproduce the enthalpies of $Co^{2+}$-MBP interaction over the whole $Co^{2+}$ concentrations. The solvation parameters recovered from the solvation model were attributed to the structural change of MBP due to the metal ion interaction. It was found that there is a set of three identical and noninteracting binding sites for $Co^{2+}$ ions. The association equilibrium constant is 0.015 ${\mu}M^{-1}$. The molar enthalpy of binding is $\Delta$H = −14.60 kJ $mol^{-1}$.

• Bulletin of the Korean Chemical Society
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• v.35 no.4
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• pp.1087-1090
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• 2014

A simple and general method that selectively introduces metal binding sites into a protein can greatly increase the ability to design and biosynthesize artificial metalloproteins. Here, we report the incorporation of a phenanthroline-containing amino acid (Phen-Ala) into proteins in Escherichia coli by using the $tRNA{^{Tyr}}_{CUA}$ and tyrosyl aminoacyl-tRNA synthetase pair (BpyRS) from Methanococcus jannaschii, which was originally developed for a bipyridine-containing amino acid (Bpy-Ala). The incorporation efficiency of BpyRS for Phen-Ala was comparable to that for Bpy-Ala. Because of its high metal-binding ability and characteristic spectral properties, Phen-Ala can be a useful alternative to the existing metal-chelating amino acids for the design and synthesis of artificial metalloproteins.

• Bulletin of the Korean Chemical Society
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• v.32 no.12
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• pp.4337-4340
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• 2011

The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain of Escherichia coli harboring an induction controller plasmid. The fusion protein was bound to the immobilized metal ions, and the denaturation and renaturation of the fusion protein were performed as a part of the purification procedure. After the elution of the fusion protein, the peptide hormone was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The purity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 5 and 2 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this peptide will enable structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.

• BMB Reports
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• v.40 no.5
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• pp.649-655
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• 2007

The nsp14 protein is an exoribonuclease that is encoded by severe acute respiratory syndrome coronavirus (SARS-CoV). We have cloned and expressed the nsp14 protein in Escherichia coli, and characterized the nature and the role(s) of the metal ions in the reaction chemistry. The purified recombinant nsp14 protein digested a 5'-labeled RNA molecule, but failed to digest the RNA substrate that is modified with fluorescein group at the 3'-hydroxyl group, suggesting a 3'-to-5' exoribonuclease activity. The exoribonuclease activity requires $Mg^{2+}$ as a cofactor. Isothermal titration calorimetry (ITC) analysis indicated a two-metal binding mode for divalent cations by nsp14. Endogenous tryptophan fluorescence and circular dichroism (CD) spectra measurements showed that there was a structural change of nsp14 when binding with metal ions. We propose that the conformational change induced by metal ions may be a prerequisite for catalytic activity by correctly positioning the side chains of the residues located in the active site of the enzyme.

• Proceedings of the Korean Biophysical Society Conference
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• 2002.06b
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• pp.18-18
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• 2002

Nitrogenase, comprised of the MoFe and Fe proteins, catalyzes the reduction of dinitrogen to ammonia at ambient temperature and pressure. The MoFe protein contains two metal centers, the P-cluster (Fe8S7-8) and the FeMo-cofactor (Fe7S9：homocitrate), the substrate binding site. Despite the availability of the crystal structure of the MoFe protein, suprisingly little is known about the molecular details of catalysis at the active site, and no small-molecule substrate or inhibitor had ever been shown to directly interact with a protein-bound cluster of the functioning enzyme, until our electron-nuclear double resonance(ENDOR) study of CO-inhibited nitrogenase.(omitted)

• Journal of the Korean Magnetic Resonance Society
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• v.20 no.3
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• pp.76-81
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• 2016

Copper is an elemental ion in living organisms. CopP from Helicobacter Pylori (HpCopP) is a copper(I)-binding protein and was suggested as regulator of copper metabolism in vivo. Previously, the metal binding property of HpCopP for Ag(I), Cu(I), and Cu(II) as well as the tertiary structure of HpCopP was shown. In this study, the dynamic profiles of HpCopP depending on metal binding were studied using ${^1H}-^{15}N$ steady-state NOE analysis. The heteroNOE experiment was performed for apo-CopP or metal-bound CopP. The obtained NOE values were analyzed and compared to figure out the effect of metals on the structural flexibility of HpCopP. As a result, Ag(I) and Cu(I) ions improved the rigidity of the structure while Cu(II) ion increased the flexibility of the structure, suggesting the oxidation of the CXXC motif decreases the structural stability of HpCopP.

• Journal of Photoscience
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• v.11 no.32
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• pp.65-69
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• 2004

The mechanism of interaction of four coumarin derivatives (CDS) with bovine serum albumin (BSA) was studied using spectrofluorometric technique. It was found that the coumarin ring common to all CDS makes major contribution to interaction. Binding affinities could be related to parachor values of CDS. Stem-Volmer plots indicated the presence of static component in the quenching mechanism. Results also showed that both tryptophan residues of protein are accessible to CDS. The high magnitude of rate constant of quenching indicated that the process of energy transfer occurs by intermolecular interaction forces and thus CDS binding site is in close proximity to tryptophan residues of BSA. Binding studies in the presence of the hydrophobic probe, 8-anilino-l-naphthalein-sulfonic acid showed that there is hydrophobic interaction between CDS and the probe and they do not share common sites in BSA. Thermodynamic parameters obtained from data at different temperatures showed that the binding of CDS to BSA involve hydrophobic bonds predominantly. The effects of various metal ions on the binding of CDS with BSA were also investigated.

• Journal of the Korean Magnetic Resonance Society
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• v.19 no.2
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• pp.67-73
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• 2015

The microcystin is a cyclic heptapeptide from metabolites of cyanobacteria in the genera mycrocystis, anabaeba as a result of eutrophication. It has been known that microcystin-LR is a potent inhibitor of the catalytic subunits of protein phosphatase-1 (PP-1) as well as powerful tumor promoter. The active site of microcystin actually has two metal ions $Fe^{2+}/Zn^{2+}$ close to the nucleophilic portion of PP-1-microcystin complex. We report the isolation and purification of this microcystin-LR from cyanobacteria (blue-green algae) obtained from Daechung Dam in Chung-cheong Do, Korea. Microcystin-LR was extracted from solid-phase extraction (SPE) sample preparation using a CN cartridge. The cyanobacteria extract was purified to obtain microcystin-LR by HPLC method and identified by LC/MS. The detail structural studies that can elucidate the possible role of monovalent and divalent metal ions in PP-1-microcystin complexation were carried out by utilizing molecular dynamics. Conformational changes in metal binding for ligands were monitored by molecular dynamic computation and potential of mean force (PMF) using the method of the free energy perturbation. The microcystin-metal binding PMF simulation results exhibit that microcystin can have very stable binding free energy of -10.95 kcal/mol by adopting the $Mg^{2+}$ ion at broad geometrical distribution of $0.5{\sim}4.5{\AA}$, and show that the $K^+$ ion can form a stable metal complex rather than other monovalent alkali metal ions.