• Journal of the Korean Magnetic Resonance Society
• /
• v.19 no.1
• /
• pp.49-53
• /
• 2015

MRA1997 is a 76-residue conserved hypothetical protein of Mycobacterium tuberculosis H37Ra, one of the most pathogenic bacterial species and the causative agent of tuberculosis. In this study, the sequence-specific backbone resonance assignment of MRA1997 was performed using NMR spectroscopy. Approximately 88.3% of the total resonances could be unambiguously assigned. By analyzing deviations of the $C{\alpha}$ and $C{\beta}$ chemical shift values, the secondary structure of MRA1997 was calculated. The result revealed that secondary structure of MRA 1997 consists of one ${\alpha}$-helix and five ${\beta}$-sheets. Our structural study will be a footstone towards the characterization of the three-dimensional structure of MRA1997.

• Journal of the Korean Magnetic Resonance Society
• /
• v.21 no.2
• /
• pp.50-54
• /
• 2017

Cupin-superfamily proteins represent the most functionally diverse groups of proteins and include a huge number of functionally uncharacterized proteins. Recently, YaiE, a cupin protein from Escherichia coli has been suggested to be involved in a novel activity of pyrimidine/purine nucleoside phosphorylase (PPNP). In the present study, we achieved a complete backbone NMR assignments of YaiE, by a series of heteronuclear multidimensional NMR experiments on its [$^{13}C/^{15}N$]-enriched sample. Subsequently, secondary structure analysis using the assigned chemical shift values identified 10 obvious ${\beta}-strands$ and a tentative $3_{10}-helix$. Taken all together, the results constitute the first structural characterization of a putative PPNP cupin protein.

• Biodesign
• /
• v.5 no.4
• /
• pp.136-140
• /
• 2017

Lam6 is a member of sterol-specific ${\underline{l}ipid$ transfer proteins ${\underline{a}}nchored$ at ${\underline{m}ebrane$ contact sites (LAMs). Lam6 localizes to the ER-mitochondria contact sites by its PH-like domain and the C-terminal transmembrane helix. Here, we purified and crystallized the Lam6 PH-like domain from Saccharomyces cerevisiae. To aid crystallization of the Lam6 PH-like domain, T4 lysozyme was fused to the N-terminus of the Lam6 PH-like domain with a short dipeptide linker, GlySer. The fusion protein was crystallized under the condition of 0.1 M HEPES-HCl pH 7.0, 10% (w/v) PEG 8000, and 0.1 M $Na_3$ Citrate at 293K. X-ray diffraction data of the crystals were collected to $2.4{\AA}$ resolution using synchrotron radiation. The crystals belong to the orthorhombic space group $P2_12_12_1$ with unit cell parameters $a=59.5{\AA}$, $b=60.1{\AA}$, and $c=105.6{\AA}$. The asymmetric unit contains one T4L-Lam6 molecule with a solvent content of 58.7%. The initial attempt to solve the structure by molecular replacement using the T4 lysozyme structure was successful.

• Journal of the Korean Magnetic Resonance Society
• /
• v.26 no.4
• /
• pp.46-50
• /
• 2022

Antifreeze proteins (AFPs) bind to the ice crystals and then are able to inhibit the freezing of body fluid at subzero temperatures. Type III AFPs are categorized into three subgroups, QAE1, QAE2, and SP isoforms, based on differences in their isoelectric points. We prepared the QAE2 (AFP11) and SP (AFP6) isoforms of the notched-fin eelpout AFP and their mutant constructs and determined their temperature gradients of amide proton chemical shifts (𝚫δ/𝚫T) using NMR. The nfeAFP11 (QAE2) has the distinct 𝚫δ/𝚫T pattern of the first 3₁₀ helix compared to the QAE1 isoforms. The nfeAFP6 (SP) has the deviated 𝚫δ/𝚫T values of many residues, indicating its backbone conformational distortion. The study suggests the distortion in the H-bonding interactions and backbone conformation that is important for TH activities.

• Fisheries and Aquatic Sciences
• /
• v.25 no.1
• /
• pp.1-11
• /
• 2022

Catfish is one of the most important freshwater fish farming commodities in Indonesia. Higher catfish production can be achieved by cultivating transgenic catfish carrying the growth hormone (GH) gene of African catfish (Clarias gariepinus GH, CgGH). This research focuses on analysis of the presence of the CgGH gene in transgenic G₁, G₂, and G₃ mutiara catfish broodstock, as an indication of stable CgGH inheritance. CgGH gene was isolated using the RNeasy mini kit and RT-PCR. RT-PCR revealed amplicons measuring approximately 600 bp in transgenic G₀, G₁, G₂, and G₃ mutiara catfish. The CgGH consensus sequence similarities ranged from 93.76% to 97.06%, with four functional domain sites (somatotropin-1, somatotropin-2, four α-helix, N-glycosylation, four cysteine residues) of fish GH proteins. The functional domains of fish GH proteins are conserved in G₁, G₂, and G₃ and indicate stable exogenous GH inheritance to produce transgenic catfish strains in each generation.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.5
• /
• pp.597-604
• /
• 2014

$\small{D}$-Phenylglycine aminotransferase ($\small{D}$-PhgAT) from Pseudomonas stutzeri ST-201 is useful for enzymatic synthesis of enantiomerically pure $\small{D}$-phenylglycine. However, its low protein solubility prevents its application at high substrate concentration. With an aim to increase the protein solubility, the N-terminus of $\small{D}$-PhgAT was genetically fused with short peptides ($A_1$ ${\alpha}$-helix, $A_2$ ${\alpha}$-helix, and ALAL, which is a hybrid of $A_1$ and $A_2$) from a ferredoxin enzyme of a halophilic archaeon, Halobacterium salinarum. The fused enzymes $A_1$-$\small{D}$-PhgAT, $A_2$-$\small{D}$-PhgAT, and ALAL-$\small{D}$-PhgAT displayed a reduced pI and increased in solubility by 6.1-, 5.3-, and 8.1- fold in TEMP (pH 7.6) storage, respectively, and 5-, 4.5-, and 5.9-fold in CAPSO (pH 9.5) reaction buffers, respectively, compared with the wild-type enzyme (WT-$\small{D}$-PhgAT). In addition, all the fused $\small{D}$-PhgAT displayed higher enzymatic reaction rates than the WT-DPhgAT at all concentrations of L-glutamate monosodium salt used. The highest rate, $23.82{\pm}1.47$ mM/h, was that obtained from having ALAL-$\small{D}$-PhgAT reacted with 1,500 mM of the substrate. Moreover, the halophilic fusion significantly increased the tolerance of $\small{D}$-PhgAT in the presence of NaCl and KCl, being slightly in favor of KCl, where under the same condition at 3.5 M NaCl or KCl all halophilic-fused variants showed higher activity than WT-$\small{D}$-PhgAT.

• BMB Reports
• /
• v.37 no.6
• /
• pp.709-714
• /
• 2004

The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to $42^{\circ}C$. While 40-95% operator-binding activity was shown to be retained at 35 to $42^{\circ}C$ in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to $38^{\circ}C$, although the latter showed only 10% less binding compared to that of the former at $32^{\circ}C$. The CIts391 showed almost no binding at $42^{\circ}C$. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and $42^{\circ}C$. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at $32^{\circ}C$. Interestingly, the repressor-operator complexes preformed at $0^{\circ}C$ have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to $32^{\circ}C$ after preincubation at 42 to $52^{\circ}C$. All these data suggest that the 131st proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end.

• BMB Reports
• /
• v.34 no.4
• /
• pp.293-298
• /
• 2001

Tryptic activation of the 130-kDa Bacillus thuringiensis Cry4B $\delta$-endotoxin produced protease-resistant products of ca. 47 kDa and ca. 21 kDa. The 21-kDa fragment was identified as the N-terminal five-helix bundle (${\alpha}1-{\alpha}5$,) which is a potential candidate for membrane insertion and pore formation. In this study, we constructed the recombinant clone over-expressing this putative pore-forming (PPF) fragment as inclusion bodies in Escherichia coli. The partially purified inclusions were composed of a 23-kDa protein, which cross-reacted with Cry4B antibodies, and whose N-terminus was identical to that of the 130-kDa protein. Dissimilar to protoxin inclusions, the PPF inclusions were only soluble when the carbonate buffer, pH 9.0, was supplemented with 6 M urea. After renaturation via a stepwise dialysis, the refolded PPF protein appeared to exist as an oligomer and was structurally stable upon trypsin treatment. Unlike the 130kDa protoxin, the refolded protein was able to release entrapped glucose from liposomes, and showed comparable activity to the full-length activated toxin, although it lacks larvicidal activity These results, therefore, support the notion that the PPF fragment that consists of ${\alpha}1-{\alpha}5$ of the activated Cry4B toxin is involved in membrane pore-formation.

• BMB Reports
• /
• v.43 no.3
• /
• pp.176-181
• /
• 2010

The primary sigma factor ($\sigma^{A}$) of Staphylococcus aureus, a potential drug target, was little investigated at the structural level. Using an N-terminal histidine-tagged $\sigma^{A}$ (His-$\sigma^{A}$), here we have demonstrated that it exits as a monomer in solution, possesses multiple domains, harbors primarily $\alpha$-helix and efficiently binds to a S. aureus promoter DNA in the presence of core RNA polymerase. While both N- and C-terminal ends of His-$\sigma^{A}$ are flexible in nature, two Trp residues in its DNA binding region are buried. Upon increasing the incubation temperature from 25$^{\circ}$ to 40$^{\circ}C$, $\sim$60% of the input His-$\sigma^{A}$ was cleaved by thermolysin. Aggregation of His-$\sigma^{A}$ was also initiated rapidly at 45$^{\circ}C$. From the equilibrium unfolding experiment, the Gibbs free energy of stabilization of His-$\sigma^{A}$ was estimated to be +0.70 kcal $mol^{-1}$. The data together suggest that primary sigma factor of S. aureus is an unstable protein. Core RNA polymerase however stabilized $\sigma^{A}$ appreciably.

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