• Macromolecular Research
• /
• v.13 no.5
• /
• pp.446-452
• /
• 2005

The interactions between the surface of scaffolds and specific cells play an important role in tissue engineering applications. Some cell adhesive ligand peptides including Arg-Gly-Asp (RGD) have been grafted into polymeric scaffolds to improve specific cell attachment. In order to make cell adhesive scaffolds for tissue regeneration, biodegradable nonporous poly(L-lactic acid) (PLLA) films were prepared by using a solvent casting technique with chloroform. The hydrophobic PLLA films were surface-modified by Argon plasma treatment and in situ direct acrylic acid (AA) grafting to get hydrophilic PLLA-g-PAA. The obtained carboxylic groups of PLLA-g-PAA were coupled with the amine groups of Gly-Arg-Asp-Gly (GRDG, control) and GRGD as a ligand peptide to get PLLA-g-GRDG and PLLA-g-GRGD, respectively. The surface properties of the modified PLLA films were examined by various surface analyses. The surface structures of the PLLA films were confirmed by ATR-FTIR and ESCA, whereas the immobilized amounts of the ligand peptides were 138-145 pmol/$cm^2$. The PLLA surfaces were more hydrophilic after AA and/or RGD grafting but their surface morphologies showed still relatively smoothness. Fibroblast adhesion to the PLLA surfaces was improved in the order of PLLA control

• Molecules and Cells
• /
• v.39 no.3
• /
• pp.211-216
• /
• 2016

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of $2.6{\AA}$. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant's catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.

• Molecules and Cells
• /
• v.41 no.9
• /
• pp.842-852
• /
• 2018

Notch signaling is an evolutionarily conserved pathway and involves in the regulation of various cellular and developmental processes. Ligand binding releases the intracellular domain of Notch receptor (NICD), which interacts with DNA-bound CSL [CBF1/Su(H)/Lag-1] to activate transcription of target genes. In the absence of NICD binding, CSL down-regulates target gene expression through the recruitment of various corepressor proteins including SMRT/NCoR (silencing mediator of retinoid and thyroid receptors/nuclear receptor corepressor), SHARP (SMRT/HDAC1-associated repressor protein), and KyoT2. Structural and functional studies revealed the molecular basis of these interactions, in which NICD coactivator and corepressor proteins competitively bind to ${\beta}-trefoil$ domain (BTD) of CSL using a conserved ${\varphi}W{\varphi}P$ motif (${\varphi}$ denotes any hydrophobic residues). To date, there are conflicting ideas regarding the molecular mechanism of SMRT-mediated repression of CSL as to whether CSL-SMRT interaction is direct or indirect (via the bridge factor SHARP). To solve this issue, we mapped the CSL-binding region of SMRT and employed a 'one- plus two-hybrid system' to obtain CSL interaction-defective mutants for this region. We identified the CSL-interaction module of SMRT (CIMS; amino acid 1816-1846) as the molecular determinant of its direct interaction with CSL. Notably, CIMS contains a canonical ${\varphi}W{\varphi}P$ sequence (APIWRP, amino acids 1832-1837) and directly interacts with CSL-BTD in a mode similar to other BTD-binding corepressors. Finally, we showed that CSL-interaction motif, rather than SHARP-interaction motif, of SMRT is involved in transcriptional repression of NICD in a cell-based assay. These results strongly suggest that SMRT participates in CSL-mediated repression via direct binding to CSL.

• Korean Journal of Food Science and Technology
• /
• v.33 no.6
• /
• pp.769-773
• /
• 2001

Protein content of okara and soybean were found to be 37.3% and 42.5%, respectively by micro-Kjeldahl analysis. Solubility of okara protein in phosphate buffer (pH 8) was 10% versus soy protein of 68.4%. Insolubilization of okara protein was mostly due to disulfide bonding between cysteine residues caused by excessive heat treatment during soymilk processing: hydrophobic interactions and hydrogen bondings were involved to lesser extent. Optimum extraction temperature and time were $60^{\circ}C$ and 40 min. Typical solubility profile of soy protein disappeared for okara protein though minimum solubility of the protein was around pH 3.0. Treating okara with protease was effective in solubilizing okara protein and solubility increased to 19.2%. Optimum reaction temperature and time were $80^{\circ}C$ and 50 min, respectively. Cell wall degrading enzyme did not increase solubility of the protein, however. Through enzymatic reaction okara protein could be effectively solubilized for uses as food ingredient.

• Bulletin of the Korean Chemical Society
• /
• v.24 no.10
• /
• pp.1478-1484
• /
• 2003

CRAMP was identified from a cDNA clone derived from a mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptide. Tertiary structure of CRAMP in TFE/$H_2O$ (1 : 1, v/v) solution has been determined by NMR spectroscopy previously and consists of two amphipathic $\alpha-helices$ from Leu4 to Lys10 and from Gly16 to Leu33. These two helices are connected by a flexible region from Gly11 to Gly16. Analysis of series of fragments composed of various portion of CRAMP revealed that an 18-residue fragment with the sequence from Gly16 to Leu33 (CRAMP-18) was found to retain antibacterial activity without cytotoxicity. The effects of two Phe residues at positions 14 and 15 of CRAMP-18 on structure, antibacterial activity, and interaction with lipid membranes were investigated by $Phe^{14,15}$ ${\rightarrow}$ Ala substitution (CRAMP-18-A) in the present study. Substitution of Phe with Ala in CRAMP-18 caused a significant reduction on antibacterial and membrane-disrupting activities. Tertiary structures of CRAMP-18 in 50% TFE/$H_2O$ (1 : 1, v : v) solution shows amphipathic ${\alpha}$-helix, from $Glu^2{\;}to{\;}Leu^{18}$, while CRAMP-18-A has relatively short amphipathic ${\alpha}$-helix from $Leu^4{\;}to{\;}Ala^{15}$. These results suggest that the hydrophobic property of $Phe^{14}{\;}and{\;}Phe^15$ in CRAMP-18 is essential for its antibacterial activity, ${\alpha}$-helical structure, and interactions with phospholipid membranes.

• Journal of Ginseng Research
• /
• v.44 no.5
• /
• pp.690-696
• /
• 2020

Background: As the main metabolites of ginsenosides, 20(S, R)-protopanaxadiol [PPD(S, R)] and 20(S, R)-protopanaxatriol [PPT(S, R)] are the structural basis response to a series of pharmacological effects of their parent components. Although the estrogenicity of several ginsenosides has been confirmed, however, the underlying mechanisms of their estrogenic effects are still largely unclear. In this work, PPD(S, R) and PPT(S, R) were assessed for their ability to bind and activate human estrogen receptor α (hERα) by a combination of in vitro and in silico analysis. Methods: The recombinant hERα ligand-binding domain (hERα-LBD) was expressed in E. coli strain. The direct binding interactions of ginsenosides with hERα-LBD and their ERα agonistic potency were investigated by fluorescence polarization and reporter gene assays, respectively. Then, molecular dynamics simulations were carried out to simulate the binding modes between ginsenosides and hERα-LBD to reveal the structural basis for their agonist activities toward receptor. Results: Fluorescence polarization assay revealed that PPD(S, R) and PPT(S, R) could bind to hERα-LBD with moderate affinities. In the dual luciferase reporter assay using transiently transfected MCF-7 cells, PPD(S, R) and PPT(S, R) acted as agonists of hERα. Molecular docking results showed that these ginsenosides adopted an agonist conformation in the flexible hydrophobic ligand-binding pocket. The stereostructure of C-20 hydroxyl group and the presence of C-6 hydroxyl group exerted significant influence on the hydrogen bond network and steric hindrance, respectively. Conclusion: This work may provide insight into the chemical and pharmacological screening of novel therapeutic agents from ginsenosides.

• YAKHAK HOEJI
• /
• v.53 no.4
• /
• pp.222-227
• /
• 2009

Human topoisomerase I (Topo I) plays a pivotal role in cell replication, transcription and repair and, therefore, is an important anti-cancer target. 20S-camptothecin (CPT) is a representative Topo I inhibitor. Compounds belonging to CPT family inhibit the religation step of Topo I-DNA by binding to the DNA cleavage site. Computational docking studies with Surflex-Dock were carried out to investigate the binding modes between Topo I-DNA binary complex structure and the ligand such as 20S-CPT and 9,10-substituted 20S-CPT analogues. The docking results demonstrated that most of the compounds with $IC_{50}$ value under $0.5{\mu}M$ intercalated exactly between the -1 and +1 DNA bases, deeply toward the cleavage site. The complex was stabilized by hydrogen-bonding and hydrophobic interactions with both the enzyme and the DNA. The compounds with $IC_{50}$ value above $0.5{\mu}M$ were poorly docked and did not intercalate. In addition, the docking results confirmed the overall correlation between the $IC_{50}$ values and docking scores, indicating the possible use of the modeling for the prediction of biological activity and design of potential inhibitors.

• Animal cells and systems
• /
• v.14 no.2
• /
• pp.99-114
• /
• 2010

With the consensus sequence information of the serum glucocorticoid-induced protein kinase-1 (SGK1) phosphorylation site {R-X-R-X-X-(S/T)$\Phi$; where $\Phi$ is any hydrophobic amino acid}, we noticed that the transient receptor potential vanilloid 4 (TRPV4) cation channel, a member of the TRP vanilloid subfamily, harbors the putative SGK1 phosphorylation site (on its Ser 824). We have demonstrated that TRPV4 is an SGK1 authentic substrate protein, with the phosphorylation on the Ser 824 of TRPV4 by SGK1. Further, using TRPV4 mutants (S824A and S824D), we noted that the modification of the Ser 824 activates its $Ca^{2+}$ entry, and sensitizes the TRPV4 channel to 4-$\alpha$-phorbol 12,13-didecanoate (4-${\alpha}PDD$) or heat, simultaneously enhancing its active state. Additionally, we determined that the modification of the Ser 824 controls both its plasma membrane localization and its protein interactions with calmodulin. Thus, we have proposed herein that phosphorylation on the Ser 824 of TRPV4 is one of the control points for the regulation of its functions.

• Bulletin of the Korean Chemical Society
• /
• v.28 no.6
• /
• pp.947-952
• /
• 2007

Mycobacterium tuberculosis is a pathogen responsible for 2-3 million deaths every year worldwide. The emergence of drug-resistant and multidrug-resistant tuberculosis has increased the need to identify new antituberculosis targets. Acetohydroxy acid synthase, (AHAS, EC 2.2.1.6), an enzyme involved in branched-chain amino acid synthesis, has recently been identified as a potential anti-tuberculosis target. To assist in the search for new inhibitors and “receptor-based” design of effective inhibitors of tubercular AHAS (TbAHAS), we constructed four different structural models of TbAHAS and used one of the models as a target for virtual screening of potential inhibitors. The quality of each model was assessed stereochemically by PROCHECK and found to be reliable. Up to 89% of the amino acid residues in the structural models were located in the most favored regions of the Ramachandran plot, which indicates that the conformation of each residue in the models is good. In the models, residues at the herbicide-binding site were highly conserved across 39 AHAS sequences. The binding mode of TbAHAS with a sulfonylurea herbicide was characterized by 32 hydrophobic interactions, the majority of which were contributed by residue Trp516. The model based on the highest resolution X-ray structure of yeast AHAS was used as the target for virtual screening of a chemical database containing 8300 molecules with a heterocyclic ring. We developed a short list of molecules that were predicted to bind with high scores to TbAHAS in a conformation similar to that of sulfonylurea derivatives. Five sulfonylurea herbicides that were calculated to efficiently bind TbAHAS were experimentally verified and found to inhibit enzyme activity at micromolar concentrations. The data suggest that this time-saving and costeffective computational approach can be used to discover new TbAHAS inhibitors. The list of chemicals studied in this work is supplied to facilitate independent experimental verification of the computational approach.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.10 no.2
• /
• pp.128-135
• /
• 2005

Mixed-mode hydrophobic/ionic matrices exhibit a salt-tolerant property for adsorbing target protein from high-ionic strength feedstock, which allows the application of undiluted feedstock via an expanded bed process. In the present work, a new type of mixed-mode adsorbent designed for expanded bed adsorption, Fastline $PRO^{\circledR}$, was challenged for the capture of nattokinase from the high ionic fermentation broth of Bacillus subtilis. Two important factors, pH and ion concentration, were investigated with regard to the performance of nattokinase ad-sorption. Under initial fermentation broth conditions (pH 6.6 and conductivity of 10 mS/cm) the adsorption capacity of nattokinase with Fastline PRO was high, with a maximum capacity of 5,350 U/mL adsorbent. The elution behaviors were investigated using packed bed adsorption experiments, which demonstrated that the effective desorption of nattokinase could be achieved by effecting a pH of 9.5. The biomass pulse response experiments were carried out in order to evaluate the biomass/adsorbent interactions between Bacillus subtilis cells and Fastline PRO, and to demonstrate a stable expanded bed in the feedstock containing Bacillus subtilis cells. Finally, an EBA process, utilizing mixed-mode Fastline PRO adsorbent, was optimized to capture nattokinase directly from the fermentation broth. The purification factor reached 12.3, thereby demonstrating the advantages of the mixed-mode EBA in enzyme separation.