• 한국자기공명학회논문지
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• 제10권1호
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• pp.96-104
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• 2006

The gene coding for APG8a (At4g21980), a protein from Arabidopsis thaliana, is involved in the autophagy process. The protein is an interesting candidate for structure determination by NMR spectroscopy. Toward this end, APG8a has been produced recombinantly in Escherichia coli and typical NMR experiments such as $^{15}N-HSQC$, HNCA, HN(CO)CA, CBCA(CO)NH, HCCH-TOCSY, HNCO were performed. The backbone resonances, HN, N, CA, CB, and C' were sequence-specifically assigned, and the secondary structures including 3 $\alpha$ helices and $4\beta$ strands were deduced based on the assignments. Due to the intrinsic flexibility or the effect of the denaturant, the backbone resonances were not fully observed. Since the structure calculation by NMR data was not possible, the 3-dimensional model was built based on the sequence homology, and compared with the NMR results. The overall structure of the model could explain and complement the NMR derived secondary structures.

• 한국자기공명학회논문지
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• 제20권2호
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• pp.56-60
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• 2016

Adenylate kinase (AK) enzyme which acts as the catalyst of reversible high energy phosphorylation reaction between ATP and AMP which associate with energetic metabolism and nucleic acid synthesis and signal transmission. This enzyme has three distinct domains: Core, AMP binding domain (AMPbd) and Lid domain (LID). The primary role of AMPbd and LID is associated with conformational changes due to flexibility of two domains. Three dimensional structure of human AK1 has not been confirmed and various mutation experiments have been done to determine the active sites. In this study, AK1R138A which is changed arginine[138] of LID domain with alanine[138] was made and conducted with NMR experiments, backbone dynamics analysis and mo-lecular docking dynamic simulation to find the cause of structural change and substrate binding site. Synthetic human muscle type adenylate kinase 1 (hAK1) and its mutant (AK1R138A) were re-combinded with E. coli and expressed in M9 cell. Expressed proteins were purified and finally gained at 0.520 mM hAK1 and 0.252 mM AK1R138A. Multinuclear multidimensional NMR experiments including HNCA, HN(CO)CA, were conducted for amino acid sequence analysis and signal assignments of $^1H-^{15}N$ HSQC spectrum. Our chemical shift perturbation data is shown LID domain residues and around alanine[138] and per-turbation value(0.22ppm) of valine[179] is consid-ered as inter-communication effect with LID domain and the structural change between hAK1 and AK1R138A.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.183-183
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• 1996

Bacteriophage T4 endonuclease V initiates the repair of ultraviolet (UV)-induced pyrimidine dimer photoproducts in duplex DNA. The mechanism of DNA strand cleavage involves four sequential stens: linear diffusion along dsDNA, pyrimidine dimer-specific binding,l pyrimidine dimer-DNA glycosylase activity, and Af lyase activity. Although crystal structure is known for this enzyme, solution structure has not been yet known. In order to elucidate the solution structure of this enzyme NMR spectroscopy was used. As a basis for the NMR peak assignment of the protein, HSQC spectrum was obtained on the uniformly $\^$15/N-labeled T4 endonuclease V. Each amide peak of the spectrum were classified according to amino acid spin systems by interpreting the spectrum of $\^$15/N amino acid-specific labeled T4 endonuclease V. The assignment was mainly obtained from three-dimensional NMR spectra such as 3D NOESY-HMQC, 3D TOCSY-HMQC. These experiments were carried out will uniformly $\^$15/N-labeled sample. In order to assign tile resonance of backbon atom, triple-resonance theree-dimensional NMR experiments were also performed using double labeled($\^$15/N$\^$13/C) sample. 3D HNCA, HN(CO)CA, HNCO, HN(CA)HA spectra were recorded for this purpose. The results of assignments were used to interpret the interaction of this enzyme with DNA. HSQC spectrum was obtained for T4 endonuclease V with specific $\^$15/N-labeled amino acids that have been known for important residue in catalysis. By comparing the spectrum of enzyme*DNA complex with that of the enzyme, we could confirm the important role of some residues of Thr, Arg, Tyr in activity. The results of assignments were also used to predict the secondary structure by chemical shift index (CSI).

• Molecules and Cells
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• 제25권1호
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• pp.30-42
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• 2008

The disease-specific (dsp) region and the hypersensitive response and pathogenicity (hrp) genes, including the hrpW, $hrpN_{Ep}$, and hrpC operons have previously been sequenced in Erwinia pyrifoliae WT3 [Shrestha et al. (2005a)]. In this study, the remaining hrp genes, including the hrpC, hrpA, hrpS, hrpXY, hrpL and hrpJ operons, were determined. The hrp genes cluster (ca. 38 kb) was comprised of eight transcriptional units and contained nine hrc (hrp conserved) genes. The genetic organization of the hrp/hrc genes and their orientation for the transcriptions were also similar to and collinear with those of E. amylovora, showing ${\geq}80%$ homologies. However, ORFU1 and ORFU2 of unknown functions, present between the hrpA and hrpS operons of E. amylovora, were absent in E. pyrifoliae. To determine the HR active domains, several proteins were prepared from truncated fragments of the N-terminal and the C-terminal regions of $HrpN_{Ep}$ protein of E. pyrifoliae. The proteins prepared from the N-terminal region elicited HR, but not from those of the C-terminal region indicating that HR active domains are located in only N-terminal region of the $HrpN_{Ep}$ protein. Two synthetic oligopeptides produced HR on tobacco confirming presence of two HR active domains in the $HrpN_{Ep}$. The HR positive N-terminal fragment ($HN{\Delta}C187$) was further narrowed down by deleting C-terminal amino acids and internal amino acids to investigate whether amino acid insertion region have role in faster and stronger HR activity in $HrpN_{Ep}$ than $HrpN_{Ea}$. The $HrpN_{Ep}$ mutant proteins $HN{\Delta}C187$ (D1AIR), $HN{\Delta}C187$ (D2AIR) and $HN{\Delta}C187$ (DM41) retained similar HR activation to that of wild-type $HrpN_{Ep}$. However, the $HrpN_{Ep}$ mutant protein $HN{\Delta}C187$ (D3AIR) lacking third amino acid insertion region (102 to 113 aa) reduced HR when compared to that of wild-type $HrpN_{Ep}$. Reduction in HR elicitation could not be observed when single amino acids at different positions were substituted at third amino acids insertion region. But, substitution of amino acids at L103R, L106K and L110R showed reduction in HR activity on tobacco suggesting their importance in activation of HR faster in the $HrpN_{Ep}$ although it requires further detailed analysis.

• 자원환경지질
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• 제35권3호
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• pp.257-271
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• 2002

도시지역의 오염된 우수관퇴적물과 관련된 주요 관심사항 중의 하나는 산성비의 pH와 탄산염광물의 완충역할에 따른 중금속 원소의 단기적인 유동도이다. 탄산염광물의 완충역할은 적정법에 의해 평가되었다. 용출실험은 다양한 초기 질산농도를 갖는 용액으로 24 시간동안 실시하였다. 우수관퇴적물은 주로 방해석과 백운석에 의해 완충되는 것으로 나타났다. 탄산염광물을 많이 함유하고 있는 퇴적물 시료의 경우, pH가 산도 (acidity)의 증가에 따라 서서히 감소하였다. 탄산염광물을 덜 함유하고 있는 퇴적물 시료는 pH가 2 정도까지 급격하게 감소하다가 그 이후 천천히 감소하였다. 용출반응은 산용액이 더 첨가되어 탄산염광물의 완충역할을 완전하게 소모할 때까지 늦어졌다. pH가 감소됨에 따라 반응액의 아연, 구리, 납 및 망간 함량은 급격하게 증가하는 반면, 카드뮴, 코발트, 니켈, 크롬 및 철의 용해는 매우 느리며 제한적이었다. 중금속 원소의 용해도는 반응액의 pH값 뿐만아니라 퇴적물 입자와 수반된 금속원소의 존재형태에 의존한다. 약 산성환경에서, 아연, 카드뮴, 코발트, 니켈 및 구리의 용해작용이 점차적으로 중요해졌다. 용출실험으로부터, pH 5에서의 중금속 원소의 상대적인 유동도는 다음과 같다: 아연>카드뮴>코발트>니켈>구리>>납>크롬. 이는 약한 산성비가 오염된 우수관퇴적물로부터 아연, 카드뮴, 코발트, 니켈 및 구리를 용출시기는 반면에 납과 크롬은 용출시키지 못한다는 것을 지시한다. 방해석과 백운석 등의 탄산염광물의 완충역할은 심하게 오염된 우수관 퇴적물의 용출반응을 늦어지게 할 뿐 아니라 제한하는 데 중요한 역할을 하고 있다. 이와 간은 2차적인 환경오염은 퇴적물에 있는 금속원소가 산성비에 의해 얼마나 잘 용출되는 가에 의해 좌우된다. 물리화학적인 환경변화는 중금속에 의한 심각한 환경오염을 야기시킬 수 있다. 이런 연구결과는 오염된 퇴적물의 관리에 고려되어야 한다.