• BMB Reports
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• v.39 no.2
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• pp.150-157
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• 2006

Native grass carp (Ctenopharygodon idellus) growth hormone, has 5 cysteine amino acid residues, forms two disulphide bridges in its mature form. Recombinant grass carp growth hormone, when over-expressed in E. coli, forms inclusion bodies. In vitro oxidative renaturation of guanidine-hydrochloride dissolved recombinant grass carp growth hormone was achieved by sequential dilution and stepwise dialysis at pH 8.5. The redox potential of the refolding cocktail was maintained by glutathione disulphide/glutathione couple. The oxidative refolded protein is heterogeneous, and contains multimers, oligomers and monomers. The presence of non-disulphide-bond-forming cysteine in recombinant grass carp growth hormone enhances intermolecular disulphide bond formation and also non-native intramolecular disulphide bond formation during protein folding. The non-disulphide-bond-forming cysteine was converted to serine by PCR-mediated site-directed mutagenesis. The resulting 4-cysteine grass carp growth hormone has improved in vitro oxidative refolding properties when studied by gel filtration and reverse phase chromatography. The refolded 4-cysteine form has less hydrophobic aggregate and has only one monomeric isoform. Both refolded 4-cysteine and 5-cystiene forms are active in radioreceptor binding assay.

• BMB Reports
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• v.44 no.11
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• pp.747-752
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• 2011

To investigate the effects of disulphide bond position on the salt resistance and lipopolysaccharide (LPS)-neutralizing activity of ${\alpha}$-helical homo-dimeric antimicrobial peptides (AMPs), we synthesized an ${\alpha}$-helical model peptide ($K_6L_4W_1$) and its homo-dimeric peptides (di-$K_6L_4W_1$-N, di-$K_6L_4W_1$-M, and di-$K_6L_4W_1$-C) with a disulphide bond at the N-terminus, the central position, and the C-terminus of the molecules, respectively. Unlike $K_6L_4W_1$ and di-$K_6L_4W_1$-M, the antimicrobial activity of di-$K_6L_4W_1$-N and di-$K_6L_4W_1$-C was unaffected by 150 mM NaCl. Both di-$K_6L_4W_1$-N and di-$K_6L_4W_1$-C caused much greater inhibitory effects on nitric oxide (NO) release in LPS-induced mouse macrophage RAW 264.7 cells, compared to di-$K_6L_4W_1$-M. Taken together, our results indicate that the presence of a disulphide bond at the N- or C-terminus of the molecule, rather than at the central position, is more effective when designing salt-resistant ${\alpha}$-helical homo-dimeric AMPs with potent antimicrobial and LPS-neutralizing activities.

• Proceedings of the Korean Society of Dyers and Finishers Conference
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• 2009.03a
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• pp.69-70
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• 2009

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.133-133
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• 2003

Recently, sperm has been used as a vector to carry exogenous genes for the production of transgenic animals. However, the success in cattle is low, due to deficiencies in oocyte activation and sperm decondensation caused by high disulphide bond (S=S) content in mature sperm. This study was carried out to develop an effective method for producing transgenic animals with round spermatids (RS). Two methods of embryo production - electric fusion (EC) or intracyto-plasmic injection (IC) and three activation treatments were compared. RS were isolated from bull testes by Percoll density gradients (20, 35, 40, 45 and 90%). Fusion between ooplast and RS was performed with a single DC electric pulse (1.0 KV/cm, 45 sec) in 0.28 M mannitol solution supplemented with 100 M CaCl2 and 100 M MgCl$_2$. (중략)

• Journal of the Korean Society of Clothing and Textiles
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• v.22 no.7
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• pp.843-850
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• 1998

The purpose of this study is the investigation of chemical properties of wool treated with oxidants and protease at low temperature. The chemical degradation of the fibers were investigated by measuring $\alpha$-amimo acid contents and FT-IR analysis. In addition, urea-hydrogensulfite solubility was measured to compare to the oxidation and protease treated wool. The results were as follows. 1) By the oxidation of wool, cystine is oxidised to cysteic acid by way of the intermediate oxides, cystine-S-monooxide and cystine-S-dioxide, in the case hydrolysis catalysed by the protease catalyse. Also, $\alpha$-amimo acid contents is increased, and urea-hydrogensulfite solubility was lower than that of untreated wool. This chemical degradation of wool was occurred due to oxidate hydrolysis in the order of permonosulfate>dichloroisocyanuric acid$\geq$chlorine. 2) The chemical degradation of wool was accelerated by the protease treatment of oxidized wool. Oxidation of wool is considered to make the fiber more susceptibled to enzymatic attact by opening disulphide bond within wool. Enzymatic attact was effectively directed to the wool oxidised by permonosulfate.

• BMB Reports
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• v.37 no.3
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• pp.304-313
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• 2004

Three-dimensional (3D) models for the 65-kDa activated Cry4A and Cry4B $\delta$-endotoxins from Bacillus thuringiensis subsp. israelensis that are specifically toxic to mosquito-larvae were constructed by homology modeling, based on atomic coordinates of the Cry1Aa and Cry3Aa crystal structures. They were structurally similar to the known structures, both derived 3D models displayed a three-domain organization: the N-terminal domain (I) is a seven-helix bundle, while the middle and C-terminal domains are primarily comprise of anti-parallel $\beta$-sheets. Circular dichroism spectroscopy confirmed the secondary structural contents of the two homology-based Cry4 structures. A structural analysis of both Cry4 models revealed the following: (a) Residues Arg-235 and Arg-203 are located in the interhelical 5/6 loop within the domain I of Cry4A and Cry4B, respectively. Both are solvent exposed. This suggests that they are susceptible to tryptic cleavage. (b) The unique disulphide bond, together with a proline-rich region within the long loop connecting ${\alpha}4$ and ${\alpha}5$ of Cry4A, were identified. This implies their functional significance for membrane insertion. (c) Significant structural differences between both models were found within domain II that may reflect their different activity spectra. Structural insights from this molecular modeling study would therefore increase our understanding of the mechanic aspects of these two closely related mosquito-larvicidal proteins.

• The Journal of the Korean Society for Microbiology
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• v.12 no.1
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• pp.19-32
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• 1977

The specificity of the N- and C-terminal antigenic determinant($P_{17}$: sequence $Lys^1-{cys-}^6-Asn^{27},\;{Trp^{12}}_2-Cys^{127}-Leu^{129}$) of hen egg-white lysozyme(HL) was studied in more detail. In a Scatchard plot of the binding of $^{14}C$-acetyl HL with guinea pig purified anti-$P_{17}$ antibody experimental values bent sharply aear r=1. This suggests of two antibody populations with different affinities for HL or possible steric hindrance in the binding of a second HL molecule to the second binding site of the antibody molecule. The antigenic activities of various peptides were tested by measuring their inhibition of the binding of $^{14}C-acetyl-P_{17}$ with the antibody, Only $P_{17}$ and $P_{17}t$(sequence $Lys^1-cys^6-Homoser^{12},\;Trp^{123}-Cys^{127}-Leu^{128})$) were inhibitory, with $K_1$ values of $2.0{\times}10^4$ and $8.1{\times}10^3$, respectively. These results indicate that the direct binding site of $P_{17}$ to anti-$P_{17}$ antibody may be located in the terminal portion of $P_{17}$ (sequence $Lys^1-Cys^6-Homoser^{12},\;Trp^{123}-Cys^{127}-Leu^{129})$) while the rest of $P_{17}$ may be important in maintaining the conformation of this determinant. The single disulphide bond involved in this determinant is essential for manifestation of immunological activity.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2005.09a
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• pp.325-330
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• 2005

We are generally interested in the analysis, detection and prediction of structural motifs in proteins, in order to infer compatibility of amino acid sequence to structure in proteins of known three-dimensional structure available in the Protein Data Bank. In this context, we are analyzing some of the well-characterized structural motifs in proteins. We have analyzed simple structural motifs, such as, ${\beta}$-turns and ${\gamma}$-turns by evaluating the statistically significant type-dependent amino acid positional preferences in enlarged representative protein datasets and revised the amino acid preferences. In doing so, we identified a number of ‘unexpected’ isolated ${\beta}$-turns with a proline amino acid residue at the (i+2) position. We extended our study to the identification of multiple turns, continuous turns and to peptides that correspond to the combinations of individual ${\beta}$ and ${\gamma}$-turns in proteins and examined the hydrogen-bond interactions likely to stabilize these peptides. This led us to develop a database of structural motifs in proteins (DSMP) that would primarily allow us to make queries based on the various fields in the database for some well-characterized structural motifs, such as, helices, ${\beta}$-strands, turns, ${\beta}$-hairpins, ${\beta}$-${\alpha}$-${\beta}$, ${\psi}$-loops, ${\beta}$-sheets, disulphide bridges. We have recently implemented this information for all entries in the current PDB in a relational database called ODSMP using Oracle9i that is easy to update and maintain and added few additional structural motifs. We have also developed another relational database corresponding to amino acid sequences and their associated secondary structure for representative proteins in the PDB called PSSARD. This database allows flexible queries to be made on the compatibility of amino acid sequences in the PDB to ‘user-defined’ super-secondary structure conformation and vice-versa. Currently, we have extended this database to include nearly 23,000 protein crystal structures available in the PDB. Further, we have analyzed the ‘structural plasticity’ associated with the ${\beta}$-propeller structural motif We have developed a method to automatically detect ${\beta}$-propellers from the PDB codes. We evaluated the accuracy and consistency of predicting ${\beta}$ and ${\gamma}$-turns in proteins using the residue-coupled model. I will discuss results of our work and describe databases and software applications that have been developed.