• BMB Reports
• /
• 제40권1호
• /
• pp.22-28
• /
• 2007

MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84th amino acids) and HLH domain (98-142th amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZ$\alpha$A and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.

• 대한화학회지
• /
• 제40권6호
• /
• pp.394-400
• /
• 1996

결합성 사슬이합체를 형성할 수 있는 올리고펩티드-$(HPPHPPP)_n$-(H: 소수성 아미노산, P: 친수성 아미노산)는 온도, 수소이온 농도, 이온세기, 변성시약 등에 의해 구조적인 변화가 가능하다. 본 연구에서는 변성시약과 온도에 의한 올리고펩티드의 전이 현상을 이론적으로 고찰하였다. 사슬이합체로는 올리고펩티드20R, 변성시약으로는 구아니듐-염산을 사용하였다(20R에는 사슬 내의 정전기적 반발력이 10개가 존재하고, 사슬사이의 정전기적 반발력이 10개가 존재한다). 변성 시약에 의한 올리고펩티드의 나선에서 코일로의 전이는 급격한 것으로 보아, 변성이 일어나는 전이상태에서 올리고펩티드들은 완전한 나선구조와 무질서한 코일구조로만 되어있다. 반면에 온도에 의한 전이는 변성시약에 의한 전이보다 완만하게 일어난다. 낮은 온도에서 긴 나선 구조를 가지는 올리고펩티드가 짧은 나선 구조를 가지는 것보다 다량으로 존재한다. 온도가 증가할수록 부분적으로 변성된 분자들의 몰분율이 증가하여, 전이가 일어나는 온도에서 부분적으로 변성된 올리고펩티드가 널리 분포되어 있다.

• Molecules and Cells
• /
• 제21권2호
• /
• pp.229-236
• /
• 2006

Gaegurin 4 (GGN4), a novel peptide isolated from the skin of a Korean frog, Rana rugosa, has broad spectrum antimicrobial activity. A number of amphipathic peptides closely related to GGN4 undergo a coil to helix transition with concomitant oligomerization in lipid membranes or membrane-mimicking environments. Despite intensive study of their secondary structures, the oligomeric states of the peptides before and after the transition are not well understood. To clarify the structural basis of its antibiotic action, we used analytical ultracentrifugation to define the aggregation state of GGN4 in water, ethyl alcohol, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The maximum size of GGN4 in 15% HFIP corresponded to a decamer, whereas it was monomeric in buffer. The oligomeric transition is accompanied by a cooperative 9 nm blue-shift of maximum fluorescence emission and a large secondary structure change from an almost random coil to an ${\alpha}$-helical structure. GGN4 induces pores in lipid membranes and, using electrophysiological methods, we estimated the diameter of the pores to be exceed $7.3{\AA}$, which suggests that the minimal oligomer structure responsible is a pentamer.

• 한국전자파학회논문지
• /
• 제10권7호
• /
• pp.1095-1103
• /
• 1999

현재 helix와 monopole로 구성되는 안테나를 갖는 수납형 전화기(retractble phone)가 셀룰라 이동통신에서 가장 널리 사용되고 있다. 그러나 안테나의 monopole의 길이가 약 $\lambda$/4이므로, 강한 복사 전기장이 전화기를 사용하는 인체 두부의 외이 주변에 주로 분포한다. 이러한 전기장 분포는 두부 내에 매우 높은 국부 SAR(specific abs$\alpha$ption rate)을 야기한다. 본 논문은 이동통신단말기의 안테나 배치가 반대인 전화기 형상에 대한 두부 내 전자파 흡수율을 기존의 단말기 형상의 것과 비교, 고찰한다. 이것은 단말기 옴체의 상부가 아닌 바닥 에 안테나를 배치하는 것이다. 인체와 단말기 모델을 포함하는 계산 공간의 시간-평균 전자기장 분포를 얻기 위해 시간영역 유한차분( FDTD) 기법을 사용하였다. 그리고 SAR 분포와 국부 SAR 값이 시간-명균 전기장 분포로부터 계산되었다. 실제 상황을 고려하기 위해 해부학적 인체 두부 모델과 근사된 사용자 손이 고려되었 다. 분석된 데이터는 안테나의 이 같은 배치가 인체 두부 내에 국부 SAR을 상당히 감소시킴을 보인다.

• Molecules and Cells
• /
• 제26권2호
• /
• pp.206-211
• /
• 2008

HI0381 of Haemophilus influenzae was investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. HI0381 is a 153-residue peptidoglycan-associated outer membrane lipoprotein, and a part of the larger Tol/Pal network. Here, we report its backbone $^1H$, $^{15}N$, and $^{13}C$ resonance assignments, and secondary structure predictions. About 97% of all of the $^1HN$, $^{15}N$, $^{13}CO$, $^{13}C{\alpha}$, and $^{13}C{\beta}$ resonances covering 131 non-proline residues of the 134 residue, mature protein, were clarified by sequential and specific assignments. CSI and TALOS analyses revealed that HI0381 contains five ${\alpha}$-helices and five ${\beta}$-strands. To characterize the structure of HI0381, the effects of pH and salt concentration were investigated by CD. In addition, the structural changes occurring when HI0381 was in a membranous environment were investigated by comparing its HSQC spectra and CD data in buffer and in DPC micelles; the results showed that helix ${\alpha}4$ and strand ${\beta}4$ became aligned with the membrane. We conclude that the conformation of HI0381 is affected by the membrane environment, implying that its folded state is directly related to its function.

• 한국자기공명학회논문지
• /
• 제9권1호
• /
• pp.38-47
• /
• 2005

Axin is a scaffold protein of the APC/axin/GSK complex, binding to all of the other signalling components. Axin interacts with Glycogen synthase kinase 3$\beta$ (GSK 3$\beta$) and functions as a negative regulator of Wnt signalling pathways. To determine the solution structure of the GSK3$\beta$ binding regions of the axin, we initiated NMR study of axin fragment comprising residues 3$Val^{388} - Arg^{401}$using circular dichroism (CD) and two-dimensional NMR spectroscopy. The CD spectra of 3$axin^{pep}$ in the presence of 30% TFE displayed a standard 3$\alpha$-helical conformation, exhibiting the bound structure of 3$axin^{pep}$ to GSK3$\bata$. On the basis of experimental restraints including $NOE_s$, and $^3J_{HN\alpha} $ coupling constants, the solution conformation of $axin^{pep}$ was determined with program CNS. The 20 lowest energy structures were selected out of 50 final simulated-annealing structures in both water and TFE environment, respectively. The $RMSD_s$ for the 20 structures in TFE solution were 0.086 nm for backbone atoms and 0.195 nm for all heavy atoms, respectively. The Ramachandran plot indicates that the $\varphi$, $\psi$ angles of the 20 final structures is properly distributed in energetically acceptable regions. $Axin^pep$ in aqueous solutions consists of a stable $\alpha$-helix spanning residues form $Glu^{391}$ to $Val^{391} $, which is an interacting motif with GSK3$\beta$.

• BMB Reports
• /
• 제36권6호
• /
• pp.552-557
• /
• 2003

Oligomers with $\alpha$-aminooxy acids are reported to form very stable turn and helix structures, and they are supposed to be useful peptidomimetics for drug design. A recent report suggested that homochiral oxa-peptides form a strong eight-member-ring structure by a hydrogen bond between adjacent aminooxy-acid residues in a $CDCl_3$ solution. In order to design an $\alpha$-MSH analog with a stable turn conformation, we synthesized four tetramers and one pentamer, based on $\alpha$-MSH sequence, and determined the solution structures of the molecules by two-dimensional NMR spectroscopy and simulated annealing calculations. The solution conformations of the three peptidomimetic molecules (TLV, TDV, and TLL) in DMSO-$d_6$ contain a stable 7-membered-ring structure that is similar to a $\gamma$-turn in normal peptides. Newly-designed tetramer TDF and pentamer PDF have a ball-type rigid structure that is induced by strong hydrogen bonds between adjacent amide protons and carbonyl oxygens. In conclusion, the aminooxy acids, easily prepared from natural or unnatural amino acids, can be employed to prepare peptidomimetic analogues with well-defined turn structures for pharmaceutical interest.

• Journal of Microbiology and Biotechnology
• /
• 제24권5호
• /
• 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.

• 한국미생물·생명공학회지
• /
• 제31권2호
• /
• pp.117-123
• /
• 2003

공시균인 Saccha. erythraea IFO 13426으로부터 VB-C에 의한 erythromycin 생산 유도능이 시사된 바 있으므로, 공시균으로부터 VB-C와 특이적으로 결합하는 autoregulators 및 receptor gene을 탐색하여, EM의 생산 조절 기구를 규명하고자 하였다. 탐색의 일환으로 기존의 Streptomyce속 receptor gene의 공통배열을 primer로 이용하여 PCR을 수행하였고, 예상 크기인 120bp의 단편을 pUC19 vector에 ligation하여 E. coli DH5$\alpha$에 형질전환한 후, plasmid를 분리하여 BamHI을 처리하여 2% agarose gel에 전기영동한 결과, pUC19 (2.7kbp)외에 receptor gene PCR 산물이 120bp위치에 존재하는 것을 확인하였다. 형질전환된 plasmid로 PCR을 수행하여 염기배열을 결정한 후 해석한 결과 Streptomyces sp. 유래의 receptor gene과 유사함을 확인하였다. 따라서 Saccha. erythraea IFO 13426에는 항생물질인 erythromycin의 생산에 관여한다고 추정되는 autoregulator receptor protein을 코드하는 유전자가 존재할 것으로 예상되어 120 bp의 PCR product를 probe로 이용하여 Southern 및 colony hybridization을 통하여 3.2 kbp의 SacI 단편을 가지는 plasmid(pESG)를 제작하였고, 이를 sequencing한 결과, autoregulator receptor protein 유전자가 KpnI과 SalI을 포함하는 영역에 존재한다는 것을 알 수 있었으며 이를 EsgR이라 명명하였다. 유전자 해석 결과, EsgR은 205개의 아미노산으로 구성되어 있으며, 이는 기존의 autoregulator receptor proteins과 비교시 30%이상의 상동성을 나타내었으며, 기존의 autoregulator receptor prorein들이 하부의 항생물질 생합성 유전자들의 제어를 위해 보유하고 있는 helix-turn-helix DNA binding motif를 EsgR이 보유하고 있는 점에서, EsgR은 Saccha. erythraea가 보유하는 autoregulator receptor protein을 code하는 유전자로 추정되었다.

• Journal of Microbiology and Biotechnology
• /
• 제27권5호
• /
• pp.1023-1031
• /
• 2017

The conformational change of cellular prion protein ($PrP^C$) to its misfolded counterpart, termed $PrP^{Sc}$, is mediated by a hypothesized cellular cofactor. This cofactor is believed to interact directly with certain amino acid residues of $PrP^C$. When these are mutated into cationic amino acid residues, $PrP^{Sc}$ formation and prion replication halt in a dominant negative (DN) manner, presumably due to strong binding of the cofactor to mutated $PrP^C$, designated as DN PrP mutants. Previous studies demonstrated that plasminogen and its kringle domains bind to PrP and accelerate $PrP^{Sc}$ generation. In this study, in vitro binding analysis of kringle domains of plasminogen to Q167R DN mutant PrP (PrPQ167R) was performed in parallel with the wild type (WT) and Q218K DN mutant PrP (PrPQ218K). The binding affinity of PrPQ167R was higher than that of WT PrP, but lower than that of PrPQ218K. Scatchard analysis further indicated that, like PrPQ218K and WT PrP, PrPQ167R interaction with plasminogen occurred at multiple sites, suggesting cooperativity in this interaction. Competitive binding analysis using $\small{L}$-lysine or $\small{L}$-arginine confirmed the increase of the specificity and binding affinity of the interaction as PrP acquired DN mutations. Circular dichroism spectroscopy demonstrated that the recombinant PrPs used in this study retained the ${\alpha}$-helix-rich structure. The ${\alpha}$-helix unfolding study revealed similar conformational stability for WT and DN-mutated PrPs. This study provides an additional piece of biochemical evidence concerning the interaction of plasminogen with DN mutant PrPs.