• Journal of the Korean Magnetic Resonance Society
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• v.23 no.4
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• pp.104-107
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• 2019

Many proteins are expressed as an insoluble form during the production using Escherichia coli (E. coli) system. Although various methods are applied to increase their amounts of soluble expression, refolding is the only feasible way to obtain a target protein in some cases. Moreover, protein NMR experiments require ¹³C/¹⁵N-labeled proteins that can only be obtained from E. coli systems in terms of cost and technical difficulty. The finding of appropriate refolding conditions for a target protein is a time-consuming process. In particular, it is very difficult to determine whether the refolded protein has a native structure, when a target protein has no enzymatic activity and its refolding yield is very low. Here, we showed that 1-dimensional ¹H-¹⁵N heteronuclear single quantum correlation (1D ¹H-¹⁵N HSQC) experiment can be efficiently used to screen an optimal condition for the refolding of a target protein by monitoring both the structure and concentration of the refolded protein.

• Natural Product Sciences
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• v.12 no.4
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• pp.205-209
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• 2006

The structures of flavonoids, 2(S)-5,7-dihydroxy-8,2'-dimethoxyflavanone (1), wogonin (2), 2(S)-5,7, 2'-trihydroxy-8-methoxyflavanone (3), and 2(S)-5,2',5'-trihydroxy-7,8-dimethoxyflavanone (4), isolated from Scutellaria indica were revised on the basis of 2D NMR spectroscopy, including to gCOSY, gHSQC, and gHMBC. Compounds 1-4 were tested in vitro protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. Compounds 2 and 4 exhibited weak PTP1B inhibitory activity with $IC_{50}$ values of 208 and $337{\mu}M$, respectively.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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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).

• Applied Biological Chemistry
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• v.48 no.4
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• pp.421-424
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• 2005

The leaf of Petasites japonicus was extracted with 80% aqueous MeOH and solvent fractionated with EtOAc, n-BuOH and water successively. From the EtOAc fractions, two triterpenoids were isolated through the repeated silica gel and ODS column chromatographies. The chemical structures of the isolated terpenoids were determined as rosamutin (1) and peduncloside (2) through the interpretation of several spectral data including 2D-NMR such as $^1H-{^1H}$ gCOSY, gHSQC and gHMBC.

• Journal of the Korean Magnetic Resonance Society
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• v.3 no.2
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• pp.100-108
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• 1999

Cop protein is the transcription repressor protein in rolling circle replication plasmid. With antisense RNA, Cop protein controls the copy number of plasmid. Cop family proteins have been found in various plasmids. Among Cop family proteins, Cop studied in this paper consists of 55 amino acids (Mw. 6,400), and was known to have trimer structure. Since no structural facts are elucidated, we have carried out preliminary experiments aimed at the elucidation of its three dimensional structure. The secondary structure of Cop is studied by CD and NMR. To solve the aggregation of Cop at high concentration, we tested various detergents and salts. The addition of detergents and salts could not solve the aggregation problem. However, we found that concentration is important in solving the aggregation problem. We knew that 0.18mM in 50mM potassium phosphate without any other ingredients is maximum concentration not to aggregate. Wa also investigated the pH dependence of Cop protein, and knew that Cop protein is more stable in acid state. At various temperatures, 15N-1H HSQC spectra were measured in order to find the optimal experimental condition. To enhance the peak resolution, 3D NOESY-HSQC spectrum is acquired. Since there are NOE peaks in the NH-NH region, we knew that Cop protein has $\alpha$-helical content, which was also confirmed by CD.

• Journal of the Korean Magnetic Resonance Society
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• v.20 no.2
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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.

• Bulletin of the Korean Chemical Society
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• v.30 no.11
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• pp.2535-2541
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• 2009

Prebiotic isomaltooligosaccharide preparations contain $\alpha$-D-glucooligosaccharides comprising isomaltooligosaccharides (IMOs) and non-prebiotic maltooligosaccharides (MOs). They are both glucose oligosaccharides characterized by their degree of polymerization (DP) value (from 2 to $\sim$10), linkages types and positions (IMOs: $\alpha$-(1$\rightarrow$2, 3, 6 and in a lower proportion internal 1$\rightarrow$4) linkages, MOs: α-(1$\rightarrow$4) linkages). Their structure is the key factor for their prebiotic potential. In order to determine and elucidate the exact structure of unknown IMOs and MOs, unambiguous assignments of $^{13}C$ and $^1H$ chemical shifts of commercial standards, representative of IMOs and MOs diversity, have been determined using optimized standard one and two-dimensional experiments such as $^1H$ NMR, $^{13}C$ NMR, APT and ${^1}H-{^1}H$ COSY, TOCSY, NOESY and <$^1H-{^{13}}C$ heteronuclear HSQC, HSQC-TOCSY, and HMBC. Here we point out the differential effect of substitution by a glucose residue at different positions on chemical shifts of anomeric as well as ring carbons together with the effect of the reducing end configuration for low DP oligosaccharides and diasteroisotopic effect for H-6 protons. From this study, structural $^{13}C$ specific spectral features can be identified as tools for structural analysis of isomaltooligosaccharides.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1385-1389
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• 2007

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the formation of EPSP and inorganic phosphate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) in the biosynthesis of aromatic amino acids. To delineate the domain-specific function, we successfully isolated the discontinuous C-terminal domain (residues 1-21, linkers, 240-427) of EPSP synthase (427 residues) by site-directed mutagenesis. The engineered C-terminal domains containing no linker (CTD), or with gly-gly ($CTD^{GG}$) and gly-ser-ser-gly ($CTD^{GSSG}$) linkers were purified and characterized as having distinct native-like secondary and tertiary structures. However, isothermal titration calorimetry (ITC), $^{15}N-HSQC$,\;and\;^{31}P-NMR$ revealed that neither its substrate nor inhibitor binds the isolated domain. The isolated domain maintained structural integrity, but did not function as the half of the full-length protein.

• Natural Product Sciences
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• v.24 no.3
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• pp.189-193
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• 2018

Chemical investigation of the ethyl acetate extract of Gibberella moniliformis JS1055 endophytic fungus derived from a halophyte, Vitex rotundifolia, led to the isolation of nine compounds including 7-butyl-6,8-dihydroxy-3(R)-pent-11-enylisochroman-1-one (1), 7-butyl-6,8-dihydroxy-3(R)-pentylisochroman-1-one (2), 7-butyl6,8-dihydroxy-3(R)-pentylisochroman-1-one (3), $5{\alpha},8{\alpha}$-epidioxyergosta-6,9(11),22-trien-3-ol (4), ergosterol peroxide (5), tetradecanoic acid (6), 8-O-methylfusarubin (7), nicotinic acid (8) and adenosine (9). They were identified by extensive spectroscopic data analysis including 1D, 2D ($^1H-^1H$ COSY, HSQC, HMBC) NMR, and ESIMS. All the isolates (1-9) are reported for the first time from this endophytic fungus.

• Journal of the Korean Magnetic Resonance Society
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• v.10 no.1
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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.