• BMB Reports
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• 제38권2호
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• pp.248-252
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• 2005

Dihydrolipoamide dehydrogenase (E3) catalyzes the reoxidation of dihydrolipoyl moiety of the acyltransferase components of three $\alpha$-keto acid dehydrogenase complexes and of the hydrogen-carrier protein of the glycine cleavage system. His-457 of Pseudomonas putida E3 is suggested to interact with the hydroxyl group of Tyr-18 of the other subunit and with Glu-446, a component in the last helical structure. To examine the importance of the suggested interactions in human E3 function, the corresponding residue of human E3, Asn-473, was substituted to Leu using site-directed mutagenesis. The E3 mutant was expressed in Escherichia coli and highly purified using an affinity column. Its E3 activity was decreased about 37-fold, indicating that Asn-473 residue was important to the efficient catalytic function of human E3. Its slightly altered spectroscopic properties implied that small conformational changes could occur in the E3 mutant.

• International Journal of Industrial Entomology and Biomaterials
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• 제31권1호
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• pp.14-19
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• 2015

In a previous report, we identified several candidate antimicrobial peptides through de novo RNA sequencing of the centipede Scolopendra subspinipes mutilans. Here, we identify and characterize one of these peptides, Scolopendrasin I. We identified the centipede antimicrobial peptide Cecropin from the centipede transcriptome using an SVM algorithm, and subsequently analyzed the amino acid sequence for predicted secondary structure using a GOR algorithm. We identified an alpha helical region of Cecropin and named it Scolopendrasin I. We then assessed antimicrobial and hemolytic activity of Scolopendrasin I. Scolopendrasin I showed antimicrobial activity against various microbes, including antibiotic-resistant Gram-negative bacteria, in a radial diffusion assay. Scolopendrasin I had potent antibacterial activity against acne-associated microbes in a colony count assay and showed no hemolytic activity in a hemolysis assay. In addition, we confirmed that Scolopendrasin I bound to the surface of bacteria via a specific interaction with lipoteichoic acid and lipopolysaccharide, two components of bacterial cell membranes. In conclusion, the results presented here provide evidence that this is an efficient strategy for antimicrobial peptide candidate identification and that Scolopendrasin I has potential for successful antibiotic development.

• BMB Reports
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• 제32권6호
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• pp.561-566
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• 1999

CA(1-8)-ME(1-12) [CA-ME], composed of cecropin A(1-8) and melittin(1-12), is a synthetic antimicrobial peptide having potent antibacterial and antitumor activities with minimal hemolytic activity. In order to investigate the effects of the flexible hinge sequence, Gly-Ile-Gly, of CA-ME on antibiotic activity, CA-ME and three analogues, CA-ME1, CA-ME2, and CA-ME3, were synthesized. The Gly-Ile-Gly sequence of Ca-ME was deleted in CA-ME1 and replaced with Pro and Gly-Pro-Gly in CA-ME2 and CA-ME3, respectively. CA-ME1 and CA-ME3 showed a significant decrease in antitumor activity and phospholipid vesicle-disrupting ability. However, CA-ME2 showed similar antitumor and vesicle-disrupting activities, as compared with CA-ME. These results suggest that the flexibility or ${\beta}$-turn induced by Gly-Ile-Gly or Pro in the central part of CA-ME may be important in the electrostatic interaction of the N-terminus cationic ${\alpha}$-helical region with the cell membrane surface and the hydrophobic interaction of the C-terminus amphipathic ${\alpha}$-helical region with the hydrophobic acyl chains in the cell membrane. CA-ME3 exhibited lower antitumor and vesicle-disrupting activities than CA-ME and CA-ME2. This result suggests that the excessive ${\beta}$-turn structure caused by the Gly-Pro-Gly sequence in CA-ME3 seems to interrupt ion channel/pore formation in the lipid bilayer. We concluded that the appropriate flexibility or bilayer. We concluded that the appropriate flexibility or ${\beta}$-turn structure provided by the central hinge is responsible for the effective antibiotic activity of the antimicrobial peptides with the helix-hinge-helix structure.

• Journal of Microbiology and Biotechnology
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• 제16권6호
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• pp.880-888
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• 2006

Pleurocidin, an $\alpha$-helical cationic antimicrobial peptide, was isolated from skin mucosa of winter flounder (Pleuronectes americamus). It had strong antimicrobial activities against Gram-positive and Gram-negative bacteria, but had very weak hemolytic activity. The Gly$^{13,17}\rightarrow$Ala analog (pleurocidin-AA) showed similar antibacterial activities, but had dramatically increased hemolytic activity. The bacterial cell selectivity of pleurocidin was confirmed through the membrane-disrupting and membrane-binding affinities using dye leakage, tryptophan fluorescence blue shift, and tryptophan quenching experiments. However, the non-cell-selective antimicrobial peptide, pleurocidin-AA, interacts strongly with both negatively charged and zwitterionic phospholipid membranes, the latter of which are the major constituents of the outer leaflet of erythrocytes. Circular dihroism spectra showed that pleurocidin-AA has much higher contents of $\alpha$-helical conformation than pleurocidin. The tertiary structure determined by NMR spectroscopy showed that pleurocidin has a flexible. structure between the long helix from $Gly^3$ to $Gly^{17}$ and the short helix from $Gly^{17}$ to $Leu^{25}$. Cell-selective antimicrobial peptide pleurocidin interacts strongly with negatively charged phospholipid membranes, which mimic bacterial membranes. Structural flexibility between the two helices may play a key role in bacterial cell selectivity of pleurocidin.

• BMB Reports
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• 제47권11호
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• pp.625-630
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• 2014

Defensins, which are small cationic molecules produced by organisms as part of their innate immune response, share a common structural scaffold that is stabilized by three disulfide bridges. Coprisin is a 43-amino acid defensin-like peptide from Copris tripartitus. Here, we report the intramolecular disulfide connectivity of cysteine-rich coprisin, and show that it is the same as in other insect defensins. The disulfide bond pairings of coprisin were determined by combining the enzymatic cleavage and mass analysis. We found that the loss of any single disulfide bond in coprisin eliminated all antibacterial, but not antifungal, activity. Circular dichroism (CD) analysis showed that two disulfide bonds, Cys20-Cys39 and Cys24-Cys41, stabilize coprisin's ${\alpha}$-helical region. Moreover, a BLAST search against UniProtKB database revealed that coprisin's ${\alpha}$-helical region is highly homologous to those of other insect defensins.

• BMB Reports
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• 제35권2호
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• pp.155-164
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• 2002

The structural aspects of ervatamin B have been studied in different types of alcohol. This alcohol did not affect the structure or activity of ervatamin B under neutral conditions. At a low pH (3.0), different kinds of alcohol have different effects. Interestingly, at a certain concentration of non-fluorinated, aliphatic, monohydric alcohol, a conformational switch from the predominantly $\alpha$-helical to $\beta$-sheeted state is observed with a complete loss of tertiary structure and proteolytic activity. This is contrary to the observation that alcohol induces mostly the $\alpha$helical structure in proteins. The O-state of ervatamin B in 50% methanol at pH 3.0 has enhanced the stability towards GuHCl denaturation and shows a biphasic transition. This suggests the presence of two structural parts with different stabilities that unfold in steps. The thermal unfolding of ervatamin B in the O-state is also biphasic, which confirms the presence of two domains in the enzyme structure that unfold sequentially. The differential stabilization of the structural parts may also be a reflection of the differential stabilization of local conformations in methanol. Thermal unfolding of ervatamin B in the absence of alcohol is cooperative, both at neutral and low pH, and can be fitted to a two state model. However, at pH 2.0 the calorimetric profiles show two peaks, which indicates the presence of two structural domains in the enzyme with different thermal stabilities that are denatured more or less independently. With an increase in pH to 3.0 and 4.0, the shape of the DSC profiles change, and the two peaks converge to a predominant single peak. However, the ratio of van't Hoff enthalpy to calorimetric enthalpy is approximated to 2.0, indicating non-cooperativity in thermal unfolding.

• Bulletin of the Korean Chemical Society
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• 제27권4호
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• pp.568-572
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• 2006

To study the effect of sequence on DNA structure, the two decamer crystal structures one alternating,d(GTACGCGTAC), and the other non-alternating, d(GGCCGCGGCC), were solved. Crystals of both decamers belong to the hexagonal space group $P6_122$, with one strand in the asymmetric unit. The unit cell constants of the alternating decamer are a = b = 39.26 $\AA$, c = 77.70 $\AA$. The structure was refined with 1,828 reflections from 8.0 to 2.0 Aresolution to an R value of 21.3% with all DNA atoms and 63 water molecules. The isomorphous non-alternating decamer had unit cell dimensions of a = b = 39.05 $\AA$, c = 82.15 $\AA$. The structure was refined with 2,423 reflections from 8.0 to 2.0 $\AA$ resolution to a final R value of 22.2% for all DNA atoms and 65 water molecules. Although the average helical parameters of the decamers are typical of A-DNAs, there are some minor differences between them. The helical twist, rise, x-displacement, inclination and roll alternate in the alternating decamer, but do not in the non-alternating decamer. The backbone conformations in both structures show some differences; the residue G(7) of the alternating decamer is trans for $\alpha$ and $\gamma$ while the trans conformations are observed at the residue G(8) of the non-alternating decamer.

• Bulletin of the Korean Chemical Society
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• 제35권11호
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• pp.3267-3274
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• 2014

In order to investigate the effects of the double replacement of $\small{L}$-Pro, $\small{D}$-Pro, $\small{D}$-Leu or Nleu (the peptoid residue for Leu) in the hydrophobic face (positions 9 and 13) of amphipathic ${\alpha}$-helical non-cell-selective antimicrobial peptide $L_8K_9W_1$ on the structure, cell selectivity and mechanism of action, we synthesized a series of $L_8K_9W_1$ analogs with double replacement of $\small{L}$-Pro, $\small{D}$-Pro, $\small{D}$-Leu or Nleu in the hydrophobic face of $L_8K_9W_1$. In this study, we have confirmed that the double replacement of $\small{L}$-Pro, $\small{D}$-Pro, or Nleu in the hydrophobic face of $L_8K_9W_1$ let to a great increase in the selectivity toward bacterial cells and a complete destruction of ${\alpha}$-helical structure. Interestingly, $L_8K_9W_1$-$\small{L}$-Pro, $L_8K_9W_1$-$\small{D}$-Pro and $L_8K_9W_1$-Nleu preferentially interacted with negatively charged phospholipids, but unlike $L_8K_9W_1$ and $L_8K_9W_1$-$\small{D}$-Leu, they did not disrupt the integrity of lipid bilayers and depolarize the bacterial cytoplasmic membrane. These results suggested that the mode of action of $L_8K_9W_1$-$\small{L}$-Pro, $L_8K_9W_1$-$\small{D}$-Pro and $L_8K_9W_1$-Nleu involves the intracellular target other than the bacterial membrane. In particular, $L_8K_9W_1$-$\small{L}$-Pro, $L_8K_9W_1$-$\small{D}$-Pro and $L_8K_9W_1$-Nleu had powerful antimicrobial activity (MIC range, 1 to $4{\mu}M$) against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Taken together, our results suggested that $L_8K_9W_1$-$\small{L}$-Pro, $L_8K_9W_1$-$\small{D}$-Pro and $L_8K_9W_1$-Nleu with great cell selectivity may be promising candidates for novel therapeutic agents, complementing conventional antibiotic therapies to combat pathogenic microorganisms.

• 생명과학회지
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• 제27권5호
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• pp.584-590
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• 2017

결빙방지 단백질(Antifreeze protein, AFP)은 얼음 결정에 결합하여 결정의 성장을 억제한다. AFP는 영하의 환경에서 서식하는 생물체의 생존에 필수적이다. 겨울 넙치에서 분리된 type I AFP (AFP37)는 37 개의 잔기를 가진 ${\alpha}$-나선 구조의 펩타이드이다. 본 연구에서는 활성과 수용성이 높은 짧은 AFP 조각을 개발하고자 시도하였다. 아미노-말단 15, 21 잔기의 AFP15와 21를 설계 및 합성하였다. 이들 펩타이드의 아미노-말단에 CGG를 도입한 AFP15N and 21N을 합성하고 이황화 결합을 유도함으로써 이량체 펩타이드인dAFP15N과dAFP21N을 합성하였다. 이들의 나선 함량과 결빙방지 활성을 circular dichroism (CD) 분광법과 나노리터 삼투압계로 각각 측정하였다. 합성된 펩타이드 AFP15 AFP21, AFP15N, AFP21N, dAFP15N, dAFP21N의 나선 구조 함량은 대조구인 AFP37의 49, 41, 23.8, 28, 47.9, 51.7% 수준을 보였다. 이들의 결빙방지 활성은 AFP37의 13, 7, 0.05, 5.6, 13, 11%로 나타났다. 예상과는 달리 이량체화된 펩타이드는 단량체와 비슷한 결빙방지 활성을 보였다. 이는 이량체 펩타이드가 하나의 펩타이드로 얼음과 결합하기 보다 두 개의 개별적 펩타이드로 작용함으로써 단량체와 같은 활성을 보인 것으로 생각된다. 또한 펩타이드들에 의한 별 모양의 얼음 결정 형성은 펩타이드와 얼음의 약한 결합을 시사한다.

• Bulletin of the Korean Chemical Society
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• 제30권3호
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• pp.623-629
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• 2009

We have performed replica-exchange molecular dynamics simulations on 41 residue peptide mainly composed of NAC (non A$\beta$ component) sequence in $\alpha$-Synuclein. To investigate conformational characteristics of intrinsically unstructured peptides, we carried out structural analysis on the ‘representative structures’ for ensemble of structures occurring at different temperatures. The secondary structure profile obtained from our simulations suggests that the NAC region of $\alpha$-synuclein can be divided into roughly three helical-like segments. It is found that the overall helix-turn-helix like topology is conserved even though the conformational fluctuations grow as the temperature increases. The coordinate-based and the distance-based representative structures exhibit noticeable differences at higher temperatures while they are similar at lower temperatures. It is found that structural variations for the coordinate-based representative structures are much larger, suggesting that distance-based representative structures provide more reliable information concerning characteristic features of intrinsically unstructured proteins. The present analysis also indicates that the conformational features of representative structures at high temperatures might be related to those in membrane or low pH environment.