• BMB Reports
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• v.33 no.5
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• pp.379-384
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• 2000

The native conformation of serpins (serine protease inhibitors) is strained. Upon cleavage of the reactive center loop of serpins by a protease, the amino terminal portion of the cleaved loop is inserted into the central ${\beta}-sheet$, A sheet, as the fourth strand, with the concomitant release of the native strain. We questioned the role of protease in this conformational switch from the strained native form into a stable relaxed state. Chemical cleavage of the reactive center loop of ${\alpha}_1-antitrypsin$, a prototype serpin, using hydroxylamine dramatically increased the stability of the serpin. A circular dichroism spectrum and peptide binding study suggests that the amino terminal portion of the reactive center loop is inserted into the A sheet in the chemically-cleaved ${\alpha}_1-antitrypsin$, as in the enzymatically-cleaved molecule. These results indicate that the structural transformation of a serpin molecule does not require interaction with a protease. The results suggest that the serpin conformational switch that occurred during the complex formation with a target protease is induced by the cleavage of the reactive center loop per se.

• Molecules and Cells
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• v.25 no.1
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• pp.138-141
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• 2008

HP0892 (SwissProt/TrEMBL ID O25552) is a 90-residue conserved hypothetical protein from Helicobacter pylori strain 26695, with a calculated pI of 9.38 and a molecular mass of 10.41 kDa. It belongs to the Plasmid stabilization system protein family (PF05016) in the Pfam database. Proteins with sequence similarity to HP0892 exist in Vibrio choierae, Enterococcus faecalis, Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Escherichia coli O157. Here we report the sequence-specific backbone resonance assignments of HP0892 using multidimentional heteronuclear NMR spectroscopy. About 97.0% (422/435) of the HN, N, CO, $C{\beta}$, $C{\alpha}$ resonances of 90 residues of HP0892 were assigned. On the basis of the resonance assignments, three helical regions and four strand regions were identified using the CSI program. This study is a prerequisite for calculating the solution structure of HP0892, and will be useful for studying its interaction with other molecules.

• Journal of the Korean Magnetic Resonance Society
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• v.11 no.2
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• pp.73-84
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• 2007

MTH1821 (UniProtKB/TrEMBL ID O27849) is a 96-residue hypothetical protein from the open reading frame of Methanobacterium thermoautotrophicum H one of the target organisms of structural genomics pilot project. Proteins which contain conserved sequence compared with MTH1821 have not been discovered yet and the functional and structural information for MTH1821 is not available. Here, we present the sequence-specific backbone resonance using multidimensional heteronuc1ear NMR spectroscopy and propose the secondary structure using GetSBY software. The backbone resonances of N, HN, $C_{\alpha}$, $C_{\beta}$, CO and $H_{\alpha}$ which are necessary for a prediction of secondary structure by GetSBY were assigned about 98% (557/568). The secondary structure of MTH1821 confirmed that it is comprised of four strand regions and two helical regions. This report will provide a valuable resource for the calculation solution structure of MTH1821 and for the other hypothetical protein that is targeted for structural-based functional discovery.

• Bulletin of the Korean Chemical Society
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• v.31 no.9
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• pp.2627-2632
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• 2010

Hormone sensitive lipase (HSL) plays a major role in energy homeostasis and lipid metabolism. Several crystal structures of HSL-homolog proteins have been identified, which has led to a better understanding of its molecular function. HSL-homolog proteins exit as both monomer and dimer, but the biochemical and structural basis for such oligomeric states has not been successfully elucidated. Therefore, we determined the crystal structure of HSL-homolog protein EstE7 from a metagenome library at $2.2\;{\AA}$ resolution and characterized the oligomeric states of EstE7 both structurally and biochemically. EstE7 protein prefers the dimeric state in solution, which is supported by its higher enzymatic activity in the dimeric state. In the crystal form, EstE7 protein shows two-types of dimeric interface. Specifically, dimerization via the external ${beta}8$-strand occurred through tight association between two pseudosymmetric folds via salt bridges, hydrogen bonds and van der Waals interactions. This dimer formation was similar to that of other HSL-homolog protein structures such as AFEST, BEFA, and EstE1. We anticipate that our results will provide insight into the oligomeric state of HSL-homolog proteins.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.153-157
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• 2004

Tyrosine phenol-lyase (TPL) is a useful enzyme for the synthesis of pharmaceutical aromatic amino acids. In the current study, sequential DNA shuffling and screening were used to enhance the stability of TPL. Twenty-thousand mutants were screened, and several improved variants were isolated. One variant named A13V, in which the $13^{th}$ amino acid alanine was substituted by valine, exhibited a higher temperature and denaturant stability than the wild-type TPL. The purified mutant TPL, A13V, retained about 60% of its activity at $76^\circ{C}$, whereas the activity of the wild-type TPL decreased to less than 20% at the same temperature. Plus, A13V exhibited about 50% activity with 3 M urea, while the wild-type TPL lost almost all its catalytic activity, indicating an increased denaturant tolerance in the mutant A13V. It is speculated that the substitution of Val for the Ala in the $\beta$-strand of the N-terminal arm was responsible for the heightened stabilization, and that the current results will contribute to further research on the structural stability of TPL.

• Molecules and Cells
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• v.20 no.3
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• pp.442-445
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• 2005

HP0894 (SwissProt/TrEMBL ID O25554) is an 88-residue conserved hypothetical protein from Helicobacter pylori strain 26695 with a calculated pI of 8.5 and a molecular weight of 10.38 kDa. Proteins with sequence similarity to HP0894 exist in Vibrio choierae, Enterococcus faecalis, Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Escherichia coli O157, etc. Here we report the sequence-specific backbone resonance assignments of HP0894. About 97.5% (418/429) of the HN, N, CO, $C{\alpha}$, $C{\beta}$ resonances of the 88 residues of HP0894 were assigned. On the basis of these assignments, three helical regions and four strand regions were identified using the CSI program. This study is a prerequisite for calculating the solution structure of HP0894, and studying its interaction with its substrates, if any, and/or with other proteins.

• Journal of the Korean Applied Science and Technology
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• v.35 no.4
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• pp.1250-1259
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• 2018

In this study, Momordica charantia (MC) fermented with Leuconostoc mesenteroides (MC-LM) were assessed for the antioxidant and the antidiabetic activities. Antioxidant activities of MC and MC-LM were evaluated using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) radical. Although MC-treated groups showed little activity, 47% of activity was observed at $500{\mu}g/mL$ concentration for MC-LM and increased significantly(p<0.05) as MC-LM concentration increased. MC-LM more effectively inhibited the oxidative damage of DNA by peroxyl radical than MC and the inhibition of the strand breakage increased significantly as MC-LM concentration increased(p<0.05). Measuring the inhibition of ${\alpha}-glucosidase$ activity, which is closely related to the regulation of blood sugar, resulted in MC reduced the activity of ${\alpha}-glucosidase$ by 30% at 8 mg/mL and MC-LM at the same concentration by 60%. In addition, the effect of MC-LM on the cell viability of alloxan-treated RIN-m5F resulted in a significant increase in cell survival(p<0.05) in the group treated with MC-LM and a 20% increase in the concentration of $1000{\mu}g/mL$. As a result of insulin secretion by alloxan-treated RIN-m5F cell, the level of insulin secretion tended to increase in all group treated with MC-LM. At the concentration of $1000{\mu}g/mL$, the insulin secretion was increased by 15% in MC-LM group than in MC group. In conclusion, the results of this study suggest that fermented bitter gourd has antioxidant and antidiabetic effects.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.149-153
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• 2001

To investigate the mode of bactericidal action for antimicrobial peptide, pediocin, synthetic and mutant pediocins were prepared by direct chemical synthesis. Native pediocin was purified from Pedio-coccus acidilactici M and its conformational structure and bactericidal functions were analyzed and compared to synthetic pediocin. Schematic mode of pediocin actions, how pediocin binds on the target cell membrane, penetrates and makes tunnel are proposed. For these purposes, primary and secondary structures of pediocin was analyzed and disulfide bond assignment was also done. The pediocin purified from P. acidilactici M had high effective bactericidal ability against gram positive bacteria, especially Listeria monocytogenes and was very stable at extreme pHs and even at high temperatures such as autoclaving temperature (121$^{\circ}C$). Pediocin was consisted of 44 amino acids with four cysteines. Novel synthetic peptides were achieved by solid phase peptide synthesis(SPPS) method. To explain the function of cysteine in C-terminal region, mutant pediocin, Ped[C24A＋C44A], was synthesized and their structural and biological functions were analyzed. Second mutant pediocin, Ped[KllE], was prepared to explain the function of lysine at 11 of N-terminal part of pediocin, especially loop of $\beta$-sheet, and to predict the initial binding site of pediocin. The native and synthetic pediocins was showed random coil conformation by spectropolarimetry in moderate conditions. This conformation was observed in extreme conditions such as high temperature and low and high pHs, also. Circular dichroism(CD) data also showed the existence of $\beta$-turn structure in N-terminal part both native and synthetic pediocins. A structural model for pediocin predicts that 18 amino acids in the N-terminal part of the peptide assume a three-strand $\beta$-sheet conformation. This random coil in C-terminal part of pediocin was converted to folding structure, helix structure, in nonpolar solvents such as alcohol and TFE. The disulfide bond between $^{9}$ Cys and $^{14}$ Cys was concrete and inevitable, however, evidences of disulfide bond between $^{24}$ Cys and $^{44}$ Cys was not. Data of Ped[C24A＋C44A], pediocin mutant showed that $^{44}$ Cys was required during killing the target cells but not inevitable, since Ped[C24A＋C44A] still have bactericidal activity but much less than native pediocin. Another pediocin mutant, Ped[KllE], had still bactericidal activity, was controversial to propose that positive charge like as $^{11}$ Lys in loop or hinge in bacteriocin bound or helped to binding to microorganism with electrostatic interaction between cell membrane especially teichoic acid and positive amino acid nonspecifically. The conformation of pediocin among native, synthetic and mutant pediocins did not show big difference. The conformations between oxidized and reduced pediocin were almost similar regardless of native or synthetic.

• Proceedings of the Microbiological Society of Korea Conference
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• 2005.05a
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• pp.159-161
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• 2005

Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica is a useful model system to study the hypoviral regulation of fungal gene expression. The hypovirus is known to downregulate the fungal laccase1 (lac 1), the modulation of which is tightly governed by the inositol triphosphate ($IP_3$) and calcium second messenger system in a virus-free strain. We cloned the gene cplc1 encoding a phosphatidyl inositol-specific phospholipase C (PLC), in order to better characterize the fungal gene regulation by hypovirus. Sequence analysis of the cplc1 gene indicated that the protein product contained both the X and Y domains, which are the two conserved regions found in all known PLCs, with a 133 amino acid extension between the 2nd ${\beta}$-strand and the ${\alpha}$-helix in the X domain. In addition, the gene organization appeared to be highly similar to that of a ${\delta}$ type PLC. Disruption of the cplc1 gene resulted in slow growth and produced colonies characterized by little aerial mycelia and deep orange in color. In addition, down regulation of lac1 expression was observed. However, temperature sensitivity, osmosensitivity, virulence, and other hypovirulence-associated characteristics did not differ from the wild-type strain. Functional complementation of the cplc1-null mutant with the PLC1 gene from Saccharomyces cerevisiae restored lac1 expression, which suggests that the cloned gene encodes PLC activity. The present study indicates that the cplc1 gene is required for appropriate mycelial growth, and that it regulates the lac1 expression, which is also modulated by the hypovirus. Although several PLC genes have been identified in various simple eukaryotic organisms, the deletion analysis of the cplc1 gene in this study appears to be the first report on the functional analysis of PLC in filamentous fungi.

• Journal of Oriental Neuropsychiatry
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• v.17 no.1
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• pp.1-16
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• 2006

Objective: An water extract of the Hwang-Ryun-Chung-Sim-Um (HRC) was assessed to determine the mechanisms of its antioxidant activity. In addition, the HRC was examined in vitro for the inhibitory effect on the acetylcholinesterse (AChE). Methods: The HRC exhibited a concentration-treatment; scavenging ${\alpha},{\alpha}-diphenyl-{\beta}-picrylhydrazyl$ (DPPH) radical, linoleic acid oxidation in a thiocyanate assay system, hydroxyl radical-induced DNA nicking. We investigated mRNA levels such as catalase activity, superoxide-dismutase and glutathione peroxidase. The water extract of HRC showed inhibitory effect on AChE activity. Result: The HRC extract showed dose-dependent free radical scavenging activity, including DPPH radicals and hydroxyl radicals, using different system. The HRC was also found to be effective in protecting plasmid DNA against the strand breakage induced by Hydroxyl radicals in Fenton's reaction mixture. Futhermore, catalase mRNA expression levels increased, but SOD1 and MnSOD was not expressed. HRC in a various concentration-dependent decreased AChE mRNA levels and inhibitory effect showed AChE. Conclusion: According to the above results, it is supposed that HRC is applicable to the Dementia-type of Alzheimer clinically.