• Bulletin of the Korean Chemical Society
• /
• 제31권9호
• /
• pp.2607-2612
• /
• 2010

Human tissue-type plasminogen activator (tPA) is a valuable thrombolytic agent used to successfully treat acute myocardial infarction, thromboembolic stroke, peripheral arterial occlusion, and venous thromboembolism. Recombinant tPA is accumulated as an inactive form in inclusion bodies of E. coli and is refolded in vitro, which is accompanied by extensive aggregation. In the present study, a tPA protease domain was expressed in an active soluble form in the cytosol of E. coli Rosetta-gami cells, which allowed disulfide bond formation and supplied the tRNA molecules required for six rarely used codons in E. coli. This strategy increased the amount of soluble protease domain protein and avoided the cumbersome refolding process. The purified protease domain not only degraded tPA substrate peptides but also formed a covalently bound complex with plasminogen activator inhibitor-1, as does full-length tPA. Soluble expression and purification of tPA domains may aid in functional analyses of this multi-domain protein, which has been implicated in many physiological and pathological processes.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제37권1호
• /
• pp.15-20
• /
• 2018

Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (ERO1s) supply oxidizing equivalent to the active centers of PDI. We previously identified and characterized the ERO1 of Bombyx mori (bERO1) as a thioredoxin-like protein that shares primary sequence homology with other ERO1s. Here we compare the reactivation of inactivated rRNase and sRNase by bERO1, and show that bERO1 and bPDI cooperatively refold denatured RNase A. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis.

• Rapid Communication in Photoscience
• /
• 제3권4호
• /
• pp.70-72
• /
• 2014

Visible-light irradiation of MeCN solution containing di(hydroxo)metallo(tetraphenyl)porphyrin complex $(tppM(OH)_2$: 1a; $M=Sb(V)^+Br^-$, 1b; $M=P(V)^+Cl^-$, 1c; M=Ge(IV)) and 2-mercaptoethanol (2-ME) as a substrate under aerated condition gave bis(2-hydroxyethyl)disulfide (2-HEDS) as an oxidative product of 2-ME. It is indicated that the oxidation of 2-ME should proceed with a photocatalytic process by 1, because the turn over number (TON) for the formation of 2-HEDS was over unit. The TON was determined to be 642 as a maximum value when 1a was used as a sensitizer. The formation of 2-HDES was extremely slow under argon atmosphere. The fluorescence of 1 was not quenched by 2-ME at all, and the free energy change (${\Delta}G$) with electron transfer (ET) from 2-ME to excited triplet state of $1(^31^*)$ was estimated as a negative value. The quenching rate constant ($k_r$) of $^31^*$ by 2-ME, obtained by the kinetics for the formation of 2-HEDS, strongly depends on ${\Delta}G$. These findings indicate that 1-sensitized oxidation was initiated by photoinduced ET from 2-ME to $^31^*$ to generate both radical cation of 2-ME ($2-ME^{+\bulle}$) and porphyrin radical anion ($1^{-\bulle}$), resulting that the formation of 2-HEDS can be proceeded by the dimerization of $2-ME^{+\bulle}$, and through a catalytic cycle due to returning to 1 by the ET from $1^{-\bulle}$ to molecular oxygen.

• 생명과학회지
• /
• 제20권6호
• /
• pp.853-860
• /
• 2010

DevSR two-component system은 Mycobacterium smegmatis의 redox sensing에 관련된 주요한 regulatory system이다. DevSR system은 DevS histidine kinase와 DevR response regulator로 구성되어 있다. 저산소 조건에서 DevS histidine kinase는 활성화되어 DevR response regulator를 인산화 시키고, 인산화된 DevR response regulator는 DevR regulon의 transcriptional activator로 작용한다. DevS의 kinase activity는 DevS의 N-terminal에 위치한 GAF domain에 존재하는 heme의 ligand-binding state에 의해 결정된다. 본 연구에서는 C-terminal kinase domain의 redox-responsive cysteine (C547)이 DevS kinase activity의 redox-dependent control과 연관이 있음을 밝혔다. 산소가 존재할 때, C547 residue 사이의 disulfide bond의 형성은 DevS kinase activity를 불활성화 시킨다. $\beta$-mercaptoethanol과 dithiothreitol과 같은 환원제를 이용하여 산화된 DevS를 환원시켰을 때, DevS kinase activity가 복원된 것이 관찰되었다. 또한, C547을 alanine으로 치환했을 때, M. smegmatis의 DevS의 sensory 기능을 부분적으로 손상되는 것이 complementation 실험을 통해 in vivo 상에서 증명되었다.

• BMB Reports
• /
• 제43권1호
• /
• pp.23-28
• /
• 2010

Binary toxin consisting of BinA and BinB from Bacillus sphaericus is toxic to mosquito larvae. BinB is responsible for specific binding to the larval gut cell membrane while BinA is crucial for toxicity. To investigate functional role of cysteine in BinB, three cysteine residues at positions 67, 161, and 241 were replaced by alanine or serine. Mutations at these positions did not affect protein production and overall structure of BinB. These cysteine residues are not involved in disulfide bond formation between BinB molecules. Mosquito-larvicidal assays revealed that C67 and C161 are essential for toxicity, whereas C241 is not. Mutations at C67 and C161 resulted in weaker BinA-BinB interaction. The loss of toxicity may be due to the reduction of interactions between BinA and BinB or BinB and its receptor. C67 and C161 could also play a part during conformational changes or internalization of the binary toxin into the target cell.

• 한국잠사학회:학술대회논문집
• /
• 한국잠사학회 2003년도 제46회 춘계 학술연구 발표회
• /
• pp.44-44
• /
• 2003

Protein disulfide isomerase (PDI) is not only an isomerase catalyzing the formation of native disulfide bond(s) of nascent peptide, but also a molecular chaperone assisting chain folding. We have already reported the structure of a cDNA (bPDl) encoding PDI from Bombyx mori and the function of PDI as foldase in assisting protein folding. (omitted)

• 한국축산식품학회지
• /
• 제38권6호
• /
• pp.1203-1215
• /
• 2018

The antioxidant effects of Allium hookeri root (AHR) were investigated by evaluating lipid and protein oxidation in meatballs during refrigerated storage at $4{\pm}1^{\circ}C$. AHR was mixed at concentrations of 0.5% (w/w, T2) and 1% (w/w, T3) with minced longissimus dorsi muscle. Meatballs containing AHR (T2 and T3) were compared to those containing 0.05% (w/w) ascorbic acid (T1) as a reference and without antioxidant as a control. The 2-thiobarbituric acid reactive substances (TBARS) value, disulfide bond formation, carbonyl contents, and volatile basic nitrogen (VBN) value of T2 were lower than those of the control during storage (p<0.05). The pH values of T2 and T3 were higher than that of the control (p<0.05). Texture profile analysis of T2 revealed a lower value compared to the control (p<0.05). Therefore, the VBN value, TBARS value, disulfide bond formation, and carbonyl content in meatball containing AHR were lower than those of the control meatball. These results indicate that AHR improves the quality of meat products and functions as an antioxidant.

• Bulletin of the Korean Chemical Society
• /
• 제27권7호
• /
• pp.1048-1052
• /
• 2006

Ab initio calculations of the structure, atomic charges and natural bond orbital (NBO) have been performed at HF/6-311G** and B3LYP/6-311G** levels for the title compound of dibenzothiazyl-disulfide. The calculated results show that the two nitrogen atoms have the biggest negative charges and they are the potential sites to react with the metallic ions, which make the title compound become a di-dentate ligand. Vibrational frequencies of the title compound have been obtained and compared with the experimental value and the comparison indicates that B3LYP/6-311G** level is better than HF/6-311G** level to predict the vibrational frequencies for the system studied here. For the title compound, electronic absorption spectra calculated by time?ependent density functional theory (TD-DFT) are more accurate than Hartree-Focksingle-excitation CI (CI-Singles) method. NBO analyses show that the electronic transitions are mainly derived from the contribution of bands $\pi\rightarrow\pi^{*}$. Thermodynamic calculated results show that the formation of the title compound from 2-mercaptobenzothiazole is a spontaneous process at room temperature with the change of free Gibbs being negative value.

• 한국자기공명학회논문지
• /
• 제19권3호
• /
• pp.99-105
• /
• 2015

Hsp33 is a prokaryotic molecular chaperon that exerts a holdase activity upon response to an oxidative stress at raised temperature. In particular, intramolecular disulfide bond formation between the four conserved cysteines that bind a zinc ion in reduced state is known to be critically associated with the redox sensing. Here we report the backbone NMR assignment results of the half-oxidized Hsp33, where only two of the four cysteines form an intramolecular disulfide bond. Almost all of the resolved peaks could be unambiguously assigned, although the total assignments extent reached just about 50%. Majority of the missing assignments could be attributed to a significant spectral collapse, largely due to the oxidation-induced unfolding of the C-terminal redox-switch domain. These results support two previous suggestions: conformational change in the first oxidation step is localized mainly in the C-terminal zinc-binding domain, and the half-oxidized form would be still inactive. However, some additional regions appeared to be potentially changed from the reduced state, which suggest that the half-oxidized conformation would be an intermediate state that is more labile to heat and/or further oxidation.

• Journal of Microbiology and Biotechnology
• /
• 제16권7호
• /
• pp.1097-1103
• /
• 2006

The divalent antibody-toxins are expected to have increased binding avidities to target cells because of the two cell-binding domains. However, previous studies showed that the refolding yield of divalent antibody-toxin is very low, and it is assumed that homodimer formation of antibody-toxin is strongly interfered by the repulsion between the two large toxin domains that come close to each other during dimer formation. In this study, B3 antibody was used as a model antibody, and its Fab domain was used to construct three different kinds of Fab divalent molecules, $[B3(Fab)-toxin]_{2}$. The monomer Fab-toxin molecules were made by fusing the Fab domain of monoclonal antibody B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE), and a connecting sequence that contained spacer amino acid sequence (G4S)n (n=l, 2, 3) was inserted between Fab and PE38. The prepared divalent molecules were $[Fab-S\;1,\;2,\;3-PE38]_{2}\;(=[Fab-SKPCIST-KAS(G_{4}S)nGGPE-PE38]_{2}\;(n=1,\;2,\;3))$, and they are derivatives of previously studied $[Fab-H2cys-PE38]_{2}\;(=[Fab-SKPCIST-KASGGPE-PE38]_{2})$. In $[Fab-Sl,\;2,\;3-PE38]_{2}$, two Fab-S1, 2, 3-PE38 monomers were covalently linked by the disulfide bond bridge made from cysteine in the -SKPCIST- sequence. The insertion of spacer amino acids after the disulfide bridge resulted in a 12-18 fold higher yield of dimer formation than previously constructed $[Fab-Hlcys-PZ38]_{2}[7]$, 3-4-fold higher than $[Fab-ext-PZ38]_{2}[25]$. These two molecules have less amino acid spacer sequence between the disulfide bridge and PE38 domain. The design of $[Fab-PE38]_{2}$ in this study gave molecules with a higher refolding yield. The results of cytotoxicity assay showed a higher cytotoxic effect of these divalent molecules than that of the monovalent scFv-PE38 molecule.