• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1434-1440
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• 2006

The stable anchorage of pyridoxal 5'-phosphate (PLP) in the active site of D-amino acid transaminase (D-AT) is crucial for the enzyme catalysis. The three-dimensional structure of D-AT revealed that Glu32 is one of the active site groups that may playa role in PLP binding. To prove the role of Glu32 in PLP stability, we firstly checked the rate of the potential rate-limiting step. The kinetic analysis showed that the rate of the ${\alpha}$-deprotonation step reduced to 26-folds in E32A mutant enzyme. Spectral analyses of the reaction of D-AT with D-serine revealed that the E32A mutant enzyme failed to stabilize the key enzyme-substrate intermediate, namely a quinonoid intermediate (E-Q). Finally, analysis of circular dichroism (CD) on the wild-type and E32A mutant enzymes showed that the optical activity of PLP in the enzyme active site was lost by the removal of the carboxylic group, proving that Glu32 is indeed involved in the cofactor anchorage. The results suggested that the electrostatic interaction network through the groups from PLP, Glu32, His47, and Arg50, which was observed from the three-dimensional structure of the enzyme, plays a crucial role in the stable anchorage of the cofactor to give necessary torsion to the plane of the cofactor-substrate complex.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2013년도 제45회 하계 정기학술대회 초록집
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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.

• 한국자기공명학회논문지
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• 제9권2호
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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.

• 대한화학회지
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• 제37권11호
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• pp.951-960
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• 1993

수용액상에서 $Hg^{2+}$에 의한 trans-[Co(3,2,3-tet)$X_2]^+$ (3,2,3-tet = 4,7-diazadecane-1,10-diamine, $X_2\;=\;Cl_2,\;(NO_2)Cl,\;Br_2,\;(NO_2)Br,\;(NO_3)_2)$ 착물의 아쿠아 반응이 연구되었다. 생성물을 확인하고 반응메카니즘을 추정하기 위하여 크로마토그래피를 사용하였고 전자흡수 스펙트럼을 측정하였다. 그 결과 네자리 리간드인 3,2,3-tet가 배위된 여러가지 $trans-[Co(3,2,3-tet)X_2]^+$ 착물은 각각 아쿠아된 trans-[Co(3,2,3-tet)$(OH_2)_2]^{3+}$ 착물을 거쳐 cis-${\beta}$-[Co(3,2,3-tet)$(OH_2)_2]^{3+}$ 착물이 생성되었다. $Hg^{2+}$에 의한 trans-$[Co(3,2,3-tet)Cl_2]^+$ 착물과 trans-[Co(3,2,3-tet)$(NO_2)Cl]^+$ 착물의 아쿠아 반응에 대한 메카니즘을 추정하기 위하여 속도론적 조사를 하였따. 그 결과 $trans-[Co(3,2,3-tet)Cl_2]^+$ 착물은 D(dissociative)-메카니즘으로 진행되었고, trans-[Co(3,2,3-tet)$(NO2_)Cl]^+ $착물은 $I_d$(interchange dissociative)-메카니즘으로 진행되었다. 그리고 입체화학적인 거동을 조사하기 위하여 라세미(R,R:S,S)3,2,3-tet 대신에 키랄성이 R,R인 3,2,3,-tet를 배위시킨 trans-$[Co(R,R-3,2,3-tet)Cl_2]^+$ 착물에 $Hg^{2+}$를 용리시켰을 때 아쿠아 반응에 대한 원편광이색성(circular dichroism) 스펙트럼을 측정하여 그 절대구조를 확인한 결과 ${\Delta}-cis-{\beta}$-[Co(R,R-3,2,3-tet)$(OH_2)_2]^{3+}$ 착물이 생성되었다.

• 대한약침학회지
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• 제19권1호
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• pp.37-44
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• 2016

Objectives: This study examined the relative efficacies of a derivative of betulinic acid (dBA) and its poly (lactide-co-glycolide) (PLGA) nano-encapsulated form in A549 lung cancer cells in vivo and in co-mutagen [sodium arsenite (SA) + benzo[a]pyrene (BaP)]-induced lung cancer in mice in vivo. Methods: dBA was loaded with PLGA nanoparticles by using the standard solvent displacement method. The sizes and morphologies of nano-dBA (NdBA) were determined by using transmission electron microscopy (TEM), and their intracellular localization was verified by using confocal microscopy. The binding and interaction of NdBA with calf thymus deoxyribonucleic acid (CT-DNA) as a target were analyzed by using conventional circular dichroism (CD) and melting temperature (Tm) profile data. Apoptotic signalling cascades in vitro and in vivo were studied by using an enzyme-linked immunosorbent assay (ELISA); the ability of NdBA to cross the blood-brain barrier (BBB) was also examined. The stage of cell cycle arrest was confirmed by using a fluorescence-activated cell-sorting (FACS) data analysis. Results: The average size of the nanoparticles was ~ 110 nm. Confocal microscopy images confirmed the presence of NdBA in the cellular cytoplasm. The bio-physical properties of dBA and NdBA ascertained from the CD and the Tm profiles revealed that NdBA had greater interaction with the target DNA than dBA did. Both dBA and NdBA arrested cell proliferation at G0/G1, NdBA showing the greater effect. NdBA also induced a greater degree of cytotoxicity in A549 cells, but it had an insignificant cytotoxic effect in normal L6 cells. The results of flow cytometric, cytogenetial and histopathological studies in mice revealed that NdBA caused less nuclear condensation and DNA damage than dBA did. TEM images showed the presence of NdBA in brain samples of NdBA fed mice, indicating its ability to cross the BBB. Conclusion: Thus, compared to dBA, NdBA appears to have greater chemoprotective potential against lung cancer.

• 한국수산과학회지
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• 제38권3호
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• pp.143-147
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• 2005

Relationship between structure and biological activity was performed using the five substance P-related peptides (SPRPs) [mammalian-SP (M-SP), cod-SP (C-SP), trout-SP (T-SP), lungfish-SP (L-SP) and Ranakinin] and four SP analogs [[$His^5$]-SP (5H-SP), [$Gly^5$]-SP (5G-SP), ($Ile^8$)-SP (8I-SP) and ($Trp^8$)-Ranakinin (8W-Ranakinin)]. The circular dichroism (CD) spectra showed that all of the peptides took an unordered structure in buffer solution and artificial liposomes. However, 8W-Ranakinin undergoes conformational changes by being transferred into neutral and acidic liposomes from an unordered structure to more ordered structure. The arterial relaxing effect of the peptides was also studied with guinea-pig aorta (GPA), As a result of the studies, L-SP was about 14-fold more potant than M-SP. The order of potency compared to $EC_{50}$ value was $L-SP{\gg}M-SP>5G-SP{\ge}8I-SP>5H-SP>T-SP$, C-SP, Ranakinin, 8W-Ranakinin.

• 한국잠사곤충학회지
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• 제48권1호
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• pp.11-15
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• 2006

천잠 견단백질을 이용한 비의류용 소재개발을 위한 기초 연구의 일환으로 천잠 가수분해 견단백질 분말을 제조하여 그 구조 및 별 특성을 살펴보고 중금속 함량을 분석하였다. 1. 천잠 고치를 가수분해하여 제조한 분말의 평균분자량은 430 정도였으며 아미노산 조성 분석 결과 일반적인 천잠 견피브로인의 조성과 유사하였다. 2. 천잠 분말의 X-선 회절 분석 결과 $2{\theta}=20.34^{\circ}$ 부근과 $31.5^{\circ}$ 부근에서 강한 회절 피크를 보였으며 원이색분석 결과 220nm의 피크와 215nm의 shoulder를 나타내어 ${\alpha}-helix$와 ${\beta}-sheet$ 구조임을 알 수 있었다. 3. 시차열분석 및 열중량분석 결과 천잠 가수분해 분말은 $250^{\circ}C$ 부근에서 열분해 흡열피크를 보였으며 $270^{\circ}C$ 부근에서 열분해에 의한 최대 중량감소가 나타났다. 4. 천잠 분말에서는 수은, 비소 등 인체 독성이 강한 중금속을 관찰할 수 없었으므로 기능성 소재 개발을 위한 재료로 천잠 가수분해 분말을 활용할 수 있을 것으로 사료된다.

• 한국어병학회지
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• 제9권1호
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• pp.65-77
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• 1996

Tachyplesin I은 투구게로부터 단리된 항균성 펩티드이다. 인지질막에 대한 tachyplesin I의 작용 메카니즘을 조사하기 위해서 tachyplesin I 및 5개의 유도체를 액상법으로 합성하였다. 합성한 5개의 유도체는 [$Phe^2$]-tachyplesin I, [$Phe^{8,13}$]-techyplesin I, S-S결합을 가지지 않는 [$Cys(Acm)^{3,7,12,16}$]-tachyplesin I 및 [$Cys(Acm)^{3,7,12,16}$]-tachyplesin I 의 단편인 7(Acm)과 10(Acm)이다. 원편광이색성 (CD) 스펙트럼에서 tachyplesin I은 완충액에서 역평행 $\beta$-구조를 취하며 산성 지질막하에서는 완충액보다 약간 불규칙적인 구조를 가진다. Carboxyfluorescein 누출 실험결과 tachyplesin은 중성 및 산성 지질막과 강하게 상호 작용 하였다. 또한 형광 실험하에서는 펩티드의 소수성 부분이 인지질막의 내부에 존재하였다. 7(Acm) 및 10(Acm) 유도체를 제외한 모든 펩티드들은 lipopolysaccharide결합에 있어 거의 유사한 활성을 나타냈었다. 따라서 지질이중막은 tachyplesin I을 안정한 $\beta$-구조로부터 덜 규칙적인 구조로 구조적 변화를 유도한다고 여겨진다.

• BMB Reports
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• 제40권3호
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• pp.315-324
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• 2007

The novel $\alpha$-amylase purified from locally isolated strain, Bacillus sp. KR-8104, (KRA) (Enzyme Microb Technol; 2005; 36: 666-671) is active in a wide range of pH. The enzyme maximum activity is at pH 4.0 and it retains 90% of activity at pH 3.5. The irreversible thermoinactivation patterns of KRA and the enzyme activity are not changed in the presence and absence of $Ca^{2+}$ and EDTA. Therefore, KRA acts as a $Ca^{2+}$-independent enzyme. Based on circular dichroism (CD) data from thermal unfolding of the enzyme recorded at 222 nm, addition of $Ca^{2+}$ and EDTA similar to its irreversible thermoinactivation, does not influence the thermal denaturation of the enzyme and its Tm. The amino acid sequence of KRA was obtained from the nucleotide sequencing of PCR products of encoding gene. The deduced amino acid sequence of the enzyme revealed a very high sequence homology to Bacillus amyloliquefaciens (BAA) (85% identity, 90% similarity) and Bacillus licheniformis $\alpha$-amylases (BLA) (81% identity, 88% similarity). To elucidate and understand these characteristics of the $\alpha$-amylase, a model of 3D structure of KRA was constructed using the crystal structure of the mutant of BLA as the platform and refined with a molecular dynamics (MD) simulation program. Interestingly enough, there is only one amino acid substitution for KRA in comparison with BLA and BAA in the region involved in the calcium-binding sites. On the other hand, there are many amino acid differences between BLA and KRA at the interface of A and B domains and around the metal triad and active site area. These alterations could have a role in stabilizing the native structure of the loop in the active site cleft and maintenance and stabilization of the putative metal triad-binding site. The amino acid differences at the active site cleft and around the catalytic residues might affect their pKa values and consequently shift its pH profile. In addition, the intrinsic fluorescence intensity of the enzyme at 350 nm does not show considerable change at pH 3.5-7.0.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
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• pp.149-153
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• 2001

To investigate the mode of bactericidal action for antimicrobial peptide, pediocin, synthetic and mutant pediocins were prepared by direct chemical synthesis. Native pediocin was purified from Pedio-coccus acidilactici M and its conformational structure and bactericidal functions were analyzed and compared to synthetic pediocin. Schematic mode of pediocin actions, how pediocin binds on the target cell membrane, penetrates and makes tunnel are proposed. For these purposes, primary and secondary structures of pediocin was analyzed and disulfide bond assignment was also done. The pediocin purified from P. acidilactici M had high effective bactericidal ability against gram positive bacteria, especially Listeria monocytogenes and was very stable at extreme pHs and even at high temperatures such as autoclaving temperature (121$^{\circ}C$). Pediocin was consisted of 44 amino acids with four cysteines. Novel synthetic peptides were achieved by solid phase peptide synthesis(SPPS) method. To explain the function of cysteine in C-terminal region, mutant pediocin, Ped[C24A＋C44A], was synthesized and their structural and biological functions were analyzed. Second mutant pediocin, Ped[KllE], was prepared to explain the function of lysine at 11 of N-terminal part of pediocin, especially loop of $\beta$-sheet, and to predict the initial binding site of pediocin. The native and synthetic pediocins was showed random coil conformation by spectropolarimetry in moderate conditions. This conformation was observed in extreme conditions such as high temperature and low and high pHs, also. Circular dichroism(CD) data also showed the existence of $\beta$-turn structure in N-terminal part both native and synthetic pediocins. A structural model for pediocin predicts that 18 amino acids in the N-terminal part of the peptide assume a three-strand $\beta$-sheet conformation. This random coil in C-terminal part of pediocin was converted to folding structure, helix structure, in nonpolar solvents such as alcohol and TFE. The disulfide bond between $^{9}$ Cys and $^{14}$ Cys was concrete and inevitable, however, evidences of disulfide bond between $^{24}$ Cys and $^{44}$ Cys was not. Data of Ped[C24A＋C44A], pediocin mutant showed that $^{44}$ Cys was required during killing the target cells but not inevitable, since Ped[C24A＋C44A] still have bactericidal activity but much less than native pediocin. Another pediocin mutant, Ped[KllE], had still bactericidal activity, was controversial to propose that positive charge like as $^{11}$ Lys in loop or hinge in bacteriocin bound or helped to binding to microorganism with electrostatic interaction between cell membrane especially teichoic acid and positive amino acid nonspecifically. The conformation of pediocin among native, synthetic and mutant pediocins did not show big difference. The conformations between oxidized and reduced pediocin were almost similar regardless of native or synthetic.