• Proceedings of the Korea Crystallographic Association Conference
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• 2003.05a
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• pp.14-14
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• 2003

We have crystallized and determined the structures o the Shank PDZ domain, alone and in complex with the synthetic C-terminal hexapeptide of GKAP protein at resolutions of 1.8Å and 2.5Å, respectively. The structure revealed the structural basis of the ligand recongition by Class I PDZ-ligand interaction. Moreover, dimeric structureof shank PDZ domain suggests that the βA strand is a common surface for dimerization of PDZ domains.

• Bulletin of the Korean Chemical Society
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• v.33 no.2
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• pp.583-588
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• 2012

Functional interaction between Drosophila orphan receptor FTZ-F1 (NR5A3) and a segmentation gene product fushi tarazu (FTZ) is crucial for regulating genes related to define the identities of alternate segmental regions in the Drosophila embryo. FTZ binding to the ligand-binding domain (LBD) of FTZ-F1 is of essence in activating its transcription process. We determined solution structures of the cofactor peptide ($FTZ^{PEP}$) derived from FTZ by NMR spectroscopy. The cofactor peptide showed a nascent helical conformation in aqueous solution, however, the helicity was increased in the presence of TFE. Furthermore, $FTZ^{PEP}$ formed ${\alpha}$-helical conformation upon FTZ-F1 binding, which provides a receptor bound structure of $FTZ^{PEP}$. The solution structure of $FTZ^{PEP}$ in the presence of FTZ-F1 displays a long stretch of the ${\alpha}$-helix with a bend in the middle of helix.

• Journal of Life Science
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• v.10 no.2
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• pp.210-217
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• 2000

A kinetic profile of the catalytic domain of stromelysin-1 (SCD) using the fluorescent peptide substrate has been determined by the stopped-flow technique. The pH profile has a pH optimum of about 5.5 with an extended shoulder above pH 7. Three pKa values, 5.0, 5.7, and 9.8 are found for the free enzyme state and two pH independent Kcat/Km values of 4.1$\times$104 M-1 s-1 and 1.4$\times$104 M-1 s-1 at low and high pH, respectively. The profile is quite different in shape with other MMP family which has been reported, having broad pH optimum with two pKa values. The substrate specificity of SCD towards fluorescent heptapeptide substrates has been also examined by thin layer chromatography. The cleavage sites of the substrates have been identified using reverse-phase HPLC method.SCD cleaves Dns-PLA↓L↓WAR and Dns-PLA↓L↓FAR at two positions. However, the Dns-PLA↓LRAR, Dns-PLE↓LFAR, adn Dns-PLSar↓LFAR are cleaved exclusively at one bond. The double cleavages of Dns-PLALWAR and Dns-PLALFAR by SCD are in marked contrast to the close structurally related matrilysin. A notable feature of SCD catalysis agrees with the structural data that the S1' pocket of SCD is deeper than that of matriysin. The differences observed between SCD and matrilysin may form the basis of understanding the structural relationships and substrate specificities of the MMP family in vivo.

• Journal of Pharmaceutical Investigation
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• v.34 no.2
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• pp.101-106
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• 2004

This work aims at examining the cellular uptake behavior of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles derivatized with a protein transduction domain (PTD) using HeLa cells. For this purpose, $Tat_{49-57}$ peptide derived from transcriptional activation (Tat) protein of HIV type-1 was covalently conjugated to the terminal end of PLGA. Nanoparticles were ten prepared with the $Tat_{49-57}-PLGA$ conjugates by a spontaneous phase inversion method. The prepared particles had a mean diameter of ca. 84 nm, as measured by dynamic light scattering. The interaction of the Tat-PLGA nanoparticles with cells was examined by using confocal laser scanning microscopy. It was found tat Tat-PLGA nanoparticles incubated with HeLa cells could efficiently translocate into cytoplasm, while plain PLGA nanoparticles showed negligible cellular uptake. In addition, even at $4^{\circ}C$ or in the presence of sodium azide significant cellular internalization of Tat-PLGA nanoparticles was still observed. These results indicate that a non-endocytotic translocation mechanism might be involved in the cellular uptake of Tat-PLGA nanoparticles.

• Genomics & Informatics
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• v.20 no.1
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• pp.6.1-6.15
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• 2022

Litorilituus sediminis is a Gram-negative, aerobic, novel bacterium under the family of Colwelliaceae, has a stunning hypothetical protein containing domain called von Hippel-Lindau that has significant tumor suppressor activity. Therefore, this study was designed to elucidate the structure and function of the biologically important hypothetical protein EMK97_00595 (QBG34344.1) using several bioinformatics tools. The functional annotation exposed that the hypothetical protein is an extracellular secretory soluble signal peptide and contains the von Hippel-Lindau (VHL; VHL beta) domain that has a significant role in tumor suppression. This domain is conserved throughout evolution, as its homologs are available in various types of the organism like mammals, insects, and nematode. The gene product of VHL has a critical regulatory activity in the ubiquitous oxygen-sensing pathway. This domain has a significant role in inhibiting cell proliferation, angiogenesis progression, kidney cancer, breast cancer, and colon cancer. At last, the current study depicts that the annotated hypothetical protein is linked with tumor suppressor activity which might be of great interest to future research in the higher organism.

• Molecules and Cells
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• v.26 no.2
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• pp.140-145
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• 2008

Expression of olive flounder hepcidin I (HepI) fused with truncated OmpA signal peptides ($OmpASP_{tr}$) as directional signals does not produce soluble fusion proteins. However, by inserting amino acid segments (xxx) varying in pI and hydrophobicity/hydrophilicity into a leader sequence containing a truncated OmpASP ($OmpASP_{tr}$) and a factor Xa cleavage site (Xa) [$OmpASP_{tr}{\mid}(xxx){\mid}Xa$], we were able in some cases to express soluble recombinant HepI. Soluble expression of the recombinant protein strongly correlated with (xxx) insertions of high pI and hydrophilicity. Therefore, we modified the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ sequence by inserting Arg and Lys into (xxx) to increase the hydrophilicity of the signal peptide region. These modifications enhanced the expression of soluble recombinant HepI. Hydropathic profile analysis of the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ HepI fusion proteins revealed that the transmembrane-like domains derived from the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ sequence were larger than the internal positively charged domain native to HepI. It should therefore be possible to overcome the obstacle of internal positively charged domains to obtain soluble expression of recombinant proteins by monitoring the hydrophilicity and hydropathic profile of the signal peptide region using a computer program.

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1169-1175
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• 2009

An antifungal chitinase, ChiCW, produced by Bacillus cereus 28-9 is effective against conidial germination of Botrytis elliptica, the causal agent of lily leaf blight. ChiCW as a modular enzyme consists of a signal peptide, a catalytic domain, a fibronectin type-III-like domain, and a chitin-binding domain. When two C-terminal domains of ChiCW were truncated, $ChiCW{\Delta}FC$ (lacking the chitin-binding domain and fibronectin type III-like domain) lost its antifungal activity. Since $ChiCW{\Delta}C$ (lacking the chitin-binding domain) could not be expressed in Escherichia coli as $ChiCW{\Delta}FC$ did, a different strategy based on protein engineering technology was designed to investigate the involvement of the chitin-binding domain of ChiCW ($ChBD_{ChiCW}$) in antifungal activity in this study. Because ChiA1 of Bacillus circulans WL-12 is a modular enzyme with a higher hydrolytic activity than ChiCW but not inhibitory to conidial germination of Bo. elliptica and the similar domain composition of ChiA1 and ChiCW, the C-terminal truncated derivatives of ChiA1 were generated and used to construct chimeric chitinases with $ChBD_{ChiCW}$. When the chitin-binding domain of ChiA1 was replaced with $ChBD_{ChiCW}$, the chimeric chitinase named ChiAAAW exhibited both high enzyme activity and antifungal activity. The results indicate that $ChBD_{ChiCW}$ may play an important role in the antifungal activity of ChiCW.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1015-1025
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• 2015

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the L-leucine-specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A_6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A_6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A_6-D-Leu-domain with the A_6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

• Bulletin of the Korean Chemical Society
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• v.34 no.4
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• pp.1120-1126
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• 2013

The syndecan family consists of four transmembrane heparan sulfate proteoglycans present in most cell types and each syndecan shares a common structure containing a heparan sulfate modified extracellular domain, a single transmembrane domain and a C-terminal cytoplasmic domain. To get a better understanding of the mechanism and function of syndecan-4 which is one of the syndecan family, it is crucial to investigate its three-dimensional structure. Unfortunately, it is difficult to prepare the peptide because it is membrane-bound protein that transverses the lipid bilayer of the cell membrane. Here, we optimize the expression, purification, and characterization of transmembrane, cytoplasmic and short extracellular domains of syndecan4 (syndecan-4 eTC). Syndecan-4 eTC was successfully obtained with high purity and yield from the M9 medium. The structural information of syndecan-4 eTC was investigated by MALDI-TOF mass (MS) spectrometry, circular dichroism (CD) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. It was confirmed that syndecan-4 eTC had an ${\alpha}$-helical multimeric structure like transmembrane domain of syndecan-4 (syndecan-4 TM) in membrane environments.

• Journal of Life Science
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• v.25 no.7
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• pp.833-838
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• 2015

Multidrug-resistant super bacterial, fungal, viral, and parasitic infections are major health threaten pathogens. However, to overcome the present healthcare situation, among the leading alternatives to current drugs are antimicrobial peptides (AMPs), which are abundantly produced via various species in nature. AMPs, small host defense proteins, are in charge of the innate immunity for the protection of multicellular organisms such as fish, amphibian, reptile, plants and animals from infection. The number of AMPs identified per year has increased steadily since the 1980s. Over 2,000 natural AMPs from bacteria, protozoa, fungi, plants, and animals have been listed into the antimicrobial peptide database (APD). The majority of these AMPs (>86%) possess 11–50 amino acids with a net charge from 0 to +7 and hydrophobic percentages between 31–70%. This report classified AMP into several categories including biological source, biological functions, peptide properties, covalent bonding pattern, and 3D structure. AMP functions not only antimicrobial activity but facilitates cell biological activity such as chemotatic activity. In addition, fibroblastic reticular cell (FRC) originated from mouse lymph node stroma induced the expression of AMP in inflammatory condition. AMP induced from FRC contained whey acidic protein (WAP) domain. It suggests that the classification of AMP will be done by protein domain.