• BMB Reports
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• v.33 no.6
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• pp.493-498
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• 2000

Aquifex pyrophilus, a hyperthermophilic bacterium, has a serine-type protease that is located at the cell wall fraction with a mature size of 43 kDa. Molecular cloning of the protease gene revealed that it has an ORF of 619 amino acids with homologous catalytic site of serine-type proteases [Choi, I.-G., Bang, W.-K., Kim, S.-H., Yu, G. Y., J. Biol. Chem. (1999), Vol. 274, pp. 881-888]. Constructs containing different regions of the protease gene, including a alanine-substituted mutant at the active site serine, were constructed, and the factors affecting the expression level of the cloned protease gene in E. coli were examined. The presence of the C-terminus hydrophobic region of the protease hindered over-expression in E. coli. Also, the proteolytic activity of the expressed protein appeared to toxic to E. coli. An inactive form that deleted both of the N-terminal signal sequence and the C-terminal polar residues was over-expressed in a soluble form, purified to homogeneity, and its thermostability examined. The purified protein showed three disulfide bonds and three free sulfhydryl group. The thermal denaturation temperature of the protein was measured around $90^{\circ}C$ using a differential scanning calorimeter and circular dichroism spectrometry. The disulfide bonds were hardly reduced in the presence of reducing agents, suggesting that these disulfide bonds were located inside of the protein surface.

• BMB Reports
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• v.40 no.1
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• pp.58-64
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• 2007

Similar to the other known structures of Bacillus thuringiensis Cry $\delta$-endotoxins, the crystal structure of the 65-kDa activated Cry4Ba toxin comprises three domains which are, from the N- to C-terminus, a bundle of $\alpha$-helices, a three-$\beta$-sheet domain, and a $\beta$-sandwich. To investigate the properties of the C-terminal domain III in isolation from the rest of the toxin, the cloned Cry4Ba-domain III was over-expressed as a 21-kDa soluble protein in Escherichia coli, which cross-reacted with anti-Cry4Ba domain III monoclonal antibody. A highly-purified domain III was obtained in a monomeric form by ion-exchange and size-exclusion FPLC. Circular dichroism spectroscopy indicated that the isolated domain III fragment distinctly exists as a $\beta$-sheet structure, corresponding to the domain III structure embodied in the Cry4Ba crystal structure. In vitro binding analysis via immuno-histochemical assay revealed that the Cry4Ba-domain III protein was able to bind to the apical microvilli of the susceptible Stegomyia aegypti larval midguts, albeit at lower-binding activity when compared with the full-length active toxin. These results demonstrate for the first time that the C-terminal domain III of the Cry4Ba mosquito-larvicidal protein, which can be isolated as a native folded monomer, conceivably participates in toxin-receptor recognition.

• Bulletin of the Korean Chemical Society
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• v.27 no.11
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• pp.1801-1808
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• 2006

The interaction between whey carrier protein $\beta$-lactoglobulin type A and B (BLG-A and -B) and 2,2'-bipyridin n-hexyl dithiocarbamato Pd(II) nitrate (BPHDC-Pd(II)), a new heavy metal complex designed for anticancer property, was investigated by fluorescence spectroscopy combined with chemometry and circular dichroism (CD) techniques. A strong fluorescence quenching reaction of BPHDC-Pd(II) to BLG-A and -B was observed. Hence, BPHDC-Pd(II) complex can be bound to both BLG-A and -B, and quench the fluorescence spectra of the proteins. The quenching constant was determined using the modified Stern-Volmer equation. The binding parameters were evaluated by fluorescence quenching method. The results of binding study provided evidences presence of two and three sets of binding sites on the BLG-B and -A, respectively, for BPHDC-Pd(II) complex. Using fluorescence spectroscopy and chemometry, the ability of BLG-A and -B to form an intermediate upon interaction with BPHDC-Pd(II) complex was assessed. CD studies displayed that under influence of different concentrations of BPHDC-Pd(II) complex, the regular secondary structure of BLG-B had no significant changes, whereas for BLG-A a transition from $\alpha$-helix to $\beta$-structure was appeared. The results for both of BLG-A and -B displayed that BPHDC-Pd(II) complex can induce a conformational transition from the native form to an intermediate state with a slightly opened conformation, which is detectable with chemometry analyses.

• Macromolecular Research
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• v.12 no.3
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• pp.276-281
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• 2004

In this study we investigated the spectral properties, including electric absorption, circular and linear dichroism (CD and LD), and fluorescence emission, of DNA in a DNA-branched polyethyleneimine (BPEI) polyplex at various polymer molecular weights (M$\_$w/) and BPEI-amine-to-DNA-phosphate ratios (N/P ratios). All BPEIs exhibited a common N/P dependence in their absorption and CD spectra. At N/P ratios ＜ 1.0, we observed some hyperchromism in the absorption spectrum, red-shifts in CD bands, and decreases in LD intensity and fluorescence intensity of intercalated ethidium. At intermediate N/P ratios, complete collapse of all spectra occurred. As the N/P ratio increased further, the polyplex dissolved in water. From its characteristic CD spectrum obtained under these conditions, we conclude that the DNA exists in a B-like form. The fluorescence and LD intensities never recovered even at high N/P ratios- which indicates that the dissolved polyplex possesses positive charges and the DNA in the polyplex is condensed despite its B-form CD spectrum. The N/P range in which the absorption and CD signals collapsed was wider when the BPEIs M$\_$w/ decreased. In the case where the BPEIs M$\_$w/ was 0.8 k, recovery of the absorption and CD spectral properties at a high N/P ratio was never achieved, which suggests that the molecular weight of the polymer plays an important role in its dissolution at a high N/P ratio.

• Journal of the Korean Magnetic Resonance Society
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• v.21 no.2
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• pp.72-77
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• 2017

${\beta}-catenin$ is a key signaling protein which regulates cell signaling and gene transcription. Abnormal activation of ${\beta}-catenin$ is linked to many cancers, particularly with colorectal cancers. Although many genetic and biological studies on $Wnt/{\beta}-catenin$ have been reported and structures of the complex between ${\beta}-catenin$ and its diverse binding partners have been published, many of them have focused on armadillo repeat domain of ${\beta}-catenin$. Both N- and C-terminal domains have been suggested to regulate interactions of ${\beta}-catenin$ with other molecules, but still little is known about the C-terminal unstructured domain. To investigate the structure of this domain, construct of C-terminus was designed and structural studies were performed using size exclusion chromatography (SEC), circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopy. We observed that not only the purified full-length construct but the purified C-terminal construct also dimerizes in solution by SEC, suggesting that this domain involves in dimerization of ${\beta}-catenin$. CD and fluorescence data indicate its flexibility and structural formation in the presence of membrane environments.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.176.2-176.2
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• 2013

Property of optical diagnostics for pulse-discharged plasma in liquid and its biological applications to proteins are investigated by making use of high voltage Marx generator. The Marx generator has been consisted of 5 stages, where each charging capacitor is 0.5 ${\mu}F$, to generate a high voltage pulse with rising time of $1{\mu}s$. We have applied an input voltage of 6 kV to the each capacitor of 0.5 ${\mu}F$. High voltage pulsed plasma has been generated inside a polycarbonate tube by a single-shot operation, where the breakdown voltage is measured to be 7 kV, current of 1.2 kA, and pulse width of ~ 1 ${\mu}s$ between the two electrodes of anode-cathode whose material is made of tungsten pin, which are immersed into the liquids. We have investigated the emitted hydrogen lines for optical diagnostics of high voltage pulsed plasma. The emission line of 656.3 nm from $H-{\alpha}$ and 486.1 nm from $H-{\beta}$ have been measured by a monochromator. If we assumed that the focused plasma regions satisfy the local thermodynamic equilibrium conditions, the electron temperature and density of the high voltage pulsed plasma in liquid could be obtained by the Stark broadening of optical emission spectroscopy. For the investigation of the influence of pulsed plasma on biological proteins, we have exposed it onto the proteins such as hemoglobin and myoglobin. The structural changes in these proteins and their analysis have also been obtained by circular dichroism (CD) and ultraviolet (UV) visible spectroscopy.

• Journal of the Korean Magnetic Resonance Society
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• v.9 no.2
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• pp.110-121
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• 2005

E.coli methionyl tRNA synthetase consist of 676 amino acids and plays a key role in initiation of protein synthesis. The native form of this enzyme is a homodimer, but the monomeric enzyme truncated approximately C-terminal 120 amino acids retains the full enzymatic activities. X-ray crystal structure of the active monomeric enzyme shows that it has two domains. The N-terminal domain is thought to be a binding site for acceptor stem of tRNA, ATP, and methionine. The C-terminal domain is mainly a-helical and makes an interaction with the anticodon of $tRNA^{Met}$. Especially it is suggested that the region of helix-loop-helix including the tryptophan residue at the position 461 may be the essential for the interaction with anticodon of $tRNA^{Met}$. In this work the structure and function of E. coli methionyl-tRNA synthetase was studied by spectroscopic method (NMR, CD, Fluorescence). The importance of tryptophan residue at the position 461 was investigated by fluorescence spectroscopy. Tryptophan 461 is expected to be an essential site for the interaction between E. coli methionyl-tRNA synthetase and E. coli $tRNA^{Met}$. Proton and heteonuclear 2-dimensional NMR spectroscopy were also used to elucidate the protein-tRNA interaction.

• Molecules and Cells
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• v.42 no.3
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• pp.262-269
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• 2019

The porcine myeloid antimicrobial peptide (PMAP), one of the cathelicidin family members, contains small cationic peptides with amphipathic properties. We used a putative lysozyme originated from the bacteriophage P22 (P22 lysozyme) as a fusion partner, which was connected to the N-terminus of the PMAP36 peptide, to markedly increase the expression levels of recombinant PMAP36. The PMAP36-P22 lysozyme fusion protein with high solubility was produced in Escherichia coli. The final purified yield was approximately 1.8 mg/L. The purified PMAP36-P22 lysozyme fusion protein exhibited antimicrobial activity against both Gram-negative and Grampositive bacteria (Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Bacillus subtilis). Furthermore, we estimated its hemolytic activity against pig erythrocytes as 6% at the high concentration ($128{\mu}M$) of the PMAP36-P22 lysozyme fusion protein. Compared with the PMAP36 peptide (12%), our fusion protein exhibited half of the hemolytic activity. Overall, our recombinant PMAP36-P22 lysozyme fusion protein sustained the antimicrobial activity with the lower hemolytic activity associated with the synthetic PMAP36 peptide. This study suggests that the PMAP36-P22 lysozyme fusion system could be a crucial addition to the plethora of novel antimicrobials.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.10
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• pp.1611-1620
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• 2019

Objective: The aim was to investigate the influence of ultrasound and adenosine 5'-monophosphate (AMP) marination (UAMP) on tenderness and structure of myofibrillar proteins of beef. Methods: Five groups, the untreated meat (Control), deionized water marination (DW), ultrasound followed by DW (UDW), AMP marination (AMP), and ultrasound followed by AMP (UAMP) were studied. Myofibrillar fragmentation, cooking loss, shear force, thermograms, histological observation of meats and myofibrillar proteins properties were investigated in these different treatments. Results: The results showed that UAMP significantly increased myofibrillar fragmentation index from 152 (Control), 231 (AMP), and 307 (UDW) to 355 (p<0.05), respectively. The lowest cooking loss, shear force and peak denaturation temperature were observed in UAMP. In histological observation, UDW and UAMP had more fragmented muscular bundles than the others. Furthermore, a drastic increase in ${\alpha}$-helix and decrease in ${\beta}$-sheet of myofibrillar proteins was observed in UAMP, implying the disaggregation of protein samples. The synchronous fluorescence spectra of myofibrillar proteins in UAMP suggested the combination of ultrasound and AMP could accelerate the unfolding molecular structure and destroying hydrophobic interactions. The results of circular dichroism and synchronous fluorescence spectra for myofibrillar proteins coincided with the microstructures of beef. Conclusion: The results indicate that ultrasound combined with AMP improved meat tenderness not only by disruption in muscle integrity, increasing water retention, but also altering their spatial structure of myofibrillar proteins.

• Journal of Microbiology and Biotechnology
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• v.30 no.12
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• pp.1905-1911
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• 2020

Homoserine dehydrogenase (HSD) catalyzes the reversible conversion of ʟ-aspartate-4-semialdehyde to ʟ-homoserine in the aspartate pathway for the biosynthesis of lysine, methionine, threonine, and isoleucine. HSD has attracted great attention for medical and industrial purposes due to its recognized application in the development of pesticides and is being utilized in the large scale production of ʟ-lysine. In this study, HSD from Bacillus subtilis (BsHSD) was overexpressed in Escherichia coli and purified to homogeneity for biochemical characterization. We examined the enzymatic activity of BsHSD for ʟ-homoserine oxidation and found that BsHSD exclusively prefers NADP+ to NAD+ and that its activity was maximal at pH 9.0 and in the presence of 0.4 M NaCl. By kinetic analysis, Km values for ʟ-homoserine and NADP+ were found to be 35.08 ± 2.91 mM and 0.39 ± 0.05 mM, respectively, and the Vmax values were 2.72 ± 0.06 μmol/min-1 mg-1 and 2.79 ± 0.11 μmol/min-1 mg-1, respectively. The apparent molecular mass determined with size-exclusion chromatography indicated that BsHSD forms a tetramer, in contrast to the previously reported dimeric HSDs from other organisms. This novel oligomeric assembly can be attributed to the additional C-terminal ACT domain of BsHSD. Thermal denaturation monitoring by circular dichroism spectroscopy was used to determine its melting temperature, which was 54.8℃. The molecular and biochemical features of BsHSD revealed in this study may lay the foundation for future studies on amino acid metabolism and its application for industrial and medical purposes.