http://chineseinput.net/에서 pinyin(병음)방식으로 중국어를 변환할 수 있습니다.
변환된 중국어를 복사하여 사용하시면 됩니다.
NMR and Fluorescence Studies of DNA Binding Domain of INI1/hSNF5
Lee, Dongju,Moon, Sunjin,Yun, Jihye,Kim, Eunhee,Cheong, Chaejoon,Lee, Weontae Korean Chemical Society 2014 Bulletin of the Korean Chemical Society Vol.35 No.9
INtegrase Interactor 1 protein (INI1/hSNF5) or BRG1-associated factor 47 (BAF47) is a SWI/SNF-related matrix associated actin dependent regulator of chromatin subfamily B member. DNA binding domain of INI1/hSNF5 is cloned into E.coli expression vectors, pET32a and purified as a monomer using size exclusion chromatography. NMR data show that $INI1^{DBD}$ has folded state with high population of ${\alpha}$-helices. By fluorescence-quenching experiments, binding affinities between $INI1^{DBD}$ and two double stranded DNA fragments were determined as $29.9{\pm}2.6{\mu}M$ (GAL4_1) and $258.7{\pm}5.8$ (GAL4_2) ${\mu}M$, respectively. Our data revealed that DNA binding domain of INI1/hSNF5 binds to transcriptional DNA sequences, and it could play an important role as a transcriptional regulator.
Recombinant Expression and Purification of Cytoplasmic Domain of Syndecan-2 Proteoglycan
Weontae Lee,Young Kee Chae 대한화학회 2008 Bulletin of the Korean Chemical Society Vol.29 No.12
The cytoplasmic domain of syndecan-2, a type I transmembrane heparan sulfate proteoglycan, was overexpressed as a fused form with the ubiquitin molecule in Rosetta2(DE3)pLysS, a special strain of Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The cytoplasmic domain was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The integrity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 2 and 1.5 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this domain will enable its structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.
Lee, Chung-Kyung,Cheong, Hae-Kap,Ryu, Kyoung-Seok,Lee, Jae Il,Lee, Weontae,Jeon, Young Ho,Cheong, Chaejoon Wiley Subscription Services, Inc., A Wiley Company 2008 Proteins Vol.72 No.2
<P>Acetyl-CoA carboxylase (ACC) catalyzes the first step in fatty acid biosynthesis: the synthesis of malonyl-CoA from acetyl-CoA. As essential regulators of fatty acid biosynthesis and metabolism, ACCs are regarded as therapeutic targets for the treatment of metabolic diseases such as obesity. In ACC, the biotinoyl domain performs a critical function by transferring an activated carboxyl group from the biotin carboxylase domain to the carboxyl transferase domain, followed by carboxyl transfer to malonyl-CoA. Despite the intensive research on this enzyme, only the bacterial and yeast ACC structures are currently available. To explore the mechanism of ACC holoenzyme function, we determined the structure of the biotinoyl domain of human ACC2 and analyzed its characteristics and interaction with the biotin ligase, BirA using NMR spectroscopy. The 3D structure of the hACC2 biotinoyl domain has a similar folding topology to the earlier determined domains from E. coli and P. shermanii. However, the local structures near the biotinylation sites have notable differences that include the geometry of the consensus “Met-Lys-Met” (MKM) motif and the absence of “thumb” structure in the hACC2 biotinoyl domain. Observations of the NMR signals upon the biotinylation indicate that the biotin group of hACC2 does not affect the structure of the biotinoyl domain, while the biotin group for E. coli ACC interacts directly with the thumb residues that are not present in the hACC2 structure. These results imply that, in the E. coli ACC reaction, the biotin moiety carrying the carboxyl group from BC to CT can pause at the thumb of the BCCP domain. The human biotinoyl domain, however, lacks the thumb structure and does not have additional noncovalent interactions with the biotin moiety; thus, the flexible motion of the biotinylated lysine residue must underlie the “swinging arm” motion. The chemical shift perturbation and the cross saturation experiments of the human ACC2 holo-biotinoyl upon the addition of the biotin ligase (BirA) showed the interaction surface near the MKM motif, the two glutamic acids (Glu 926, Glu 953), and the positively charged residues (several lysine and arginine residues). This study provides insight into the mechanism of ACC holoenzyme function and supports the swinging arm model in human ACCs. Proteins 2008. © 2008 Wiley-Liss, Inc.</P>
Overexpression and Spectroscopic Characterization of Recombinant Human Tumor Suppressor p16
Lee, Weontae,Jang, Ji-Uk,Kim, Dong-Myeong,Son, Hosun,Yang, Beom-Seok The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.1
p16, which is a 16-kDa polypeptide protein, inhibits the catalytic activity of the CDK4-cyclinD complex to suppress tumor growth. Both unlabeled and isotope-labeled human tumor suppressor p16 protein were overexpressed and purified to characterize biochemical and structural properties. The purified p16 binds to monomeric GST-CDK4 and exists in a monomer conformation for several weeks at 4℃. The circular dichroism (CD) data indicates that p16 contains high percentage of α-helix and that the helix percentage maximized at pH value of 7.0. One- and two dimensional nuclear magnetic (NMR) data suggest that purified p16 from our construct has a unique folded conformation under our experimental conditions and exhibits quite stable conformational characteristics.
Lee,Weontae,Kim,Yu Sam,An,Jae Hyung,Jung,Jin-Won,Bang,Eun-Jung The Korea Science and Technology Center 1999 BMB Reports Vol.32 No.3
In order to understand the initial interaction of the substrates malonate, ATP, and CoA with malonyl-CoA synthetase, the catalytic product malonyl-CoA was characterized by NMR spectroscopy and molecular modeling. To assign proton and carbon chemical shifts, two-dimensional ¹H-¹H DQF-COSY and ¹H-¹³C HMBC experiments were used. The structure of malonyl-CoA in the solution phase was determined based on distance constraints were NOESY and ROESY spectra. The structures were well-converged around the pantetheine region with the pairwise RMSD value of 0.08nm. The solution structure exhibited a compact folded conformation with intramolecular hydrogen bonds among its carbonyl and hydroxyl groups. These findings will help us to understand the initial interaction of malonate and CoA with malonyl-CoA synthetase.
Lee, Donghan,Lee, Weontae Korean Magnetic Resonance Society 1998 Journal of the Korean Magnetic Resonance Society Vol.2 No.1
The cross peak intensities versus mixing times of 2D NOESY spectrum for a corepressor L-trp were simulated for the case of a ligand exchanging between free (AX) and bound (A'X') forms in protein/DNA complex. The direct NOE (I(AX)) of the free ligand exhibited a small positive intensity indicative of the strong dominant influence of the bound ligand. The exchange-mediated NOE peak (I(AX')) was very sensitive to corepressor exchange. However, both diagonal (I(A'A')) and direct NOE (I(A'X')) intensities of the bound ligand were not affected much at initial stage. Both peaks were severely influenced by exchange at mixing times of greater than 100 ms. In conclusion, since the NOE intensity is a function of exchange rate, the exchange effect should be considered to properly extract accurate distance information for bound ligand in the presence of conformational exchange.