47 results on '"Hyung Jin Cha"'
Search Results
2. Structure of putrescine aminotransferase from Escherichia coli provides insights into the substrate specificity among class III aminotransferases.
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Hyung Jin Cha, Jae-Hee Jeong, Catleya Rojviriya, and Yeon-Gil Kim
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Medicine ,Science - Abstract
YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.
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- 2014
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3. Purification of Therapeutic Antibodies Using the Ca2+-Dependent Phase-Transition Properties of Calsequestrin
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Heesun Park, Hyungsu Jeon, Hyung Jin Cha, Jinho Bang, Youngwoo Song, Mihyun Choi, Daekyung Sung, Won Il Choi, Jin Hyung Lee, Jae-Sung Woo, Sangyong Jon, and Sunghyun Kim
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Analytical Chemistry - Published
- 2022
4. A fluorescent nanobiosensor for the facile analysis of m6A RNA demethylase activity
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Dal-Hee Min, Hyung Jin Cha, Seo Hee Yim, Se Jin Park, Yeajee Yim, and Jae Sung Woo
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0303 health sciences ,biology ,Chemistry ,RNA Demethylase ALKBH5 ,Metals and Alloys ,RNA ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Fluorescence ,Catalysis ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,03 medical and health sciences ,Biochemistry ,Demethylase activity ,Materials Chemistry ,Ceramics and Composites ,biology.protein ,Demethylase ,Cancer development ,Quantitative analysis (chemistry) ,030304 developmental biology ,Demethylation - Abstract
RNA demethylase has recently been known to be associated with cancer development but its selective inhibitors as anti-cancer agents have rarely been investigated to date. Herein, we have developed a fluorescent nanobiosensor which enables efficient quantitative analysis of RNA demethylase ALKBH5 activity and shows a high potential for robust inhibitor screening.
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- 2020
5. Structural insights into the gating mechanism of human Cx43/GJA1 gap junction channel
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Jejoong Yoo, Hyeongseop Jeong, Hyung Jin Cha, Seu-Na Lee, Jae Sung Woo, Chang-Won Lee, Min Soo Kim, and Hyuk-Joon Lee
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Materials science ,Biophysics ,sense organs ,Gap junction channel ,Gating ,Mechanism (sociology) - Abstract
Connexin family proteins assemble into hexameric hemichannels in a cell membrane, which dock together between two adjacent membranes to form gap junction intercellular channels (GJIChs). The most ubiquitously expressed connexin Cx43 plays important roles in numerous biological processes. Here we report cryo-EM structures of Cx43 GJIChs at 3.1–3.6 Å resolutions, which show dynamic conformational changes of N-terminal helices (NTHs) caused by pH change or C-terminal truncation. Cx43 GJIChs in a channel-closing condition contain 12 protomers in gate-covering NTH (GCN) conformation, while those in opening conditions have varying compositions of GCNs and pore-lining NTHs (PLNs) resulting in various pore dimensions and electrostatic surface potentials. GCN-to-PLN transition accompanies π-helix formation in the first transmembrane helix (TM1), movement of TM2-4 that creates a side opening to the membrane, and structural stabilization of the cytoplasmic loop. Our study provides structural insights into the intercellular ion/metabolite transfer and the lateral lipid transport through Cx43 GJICh.
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- 2021
6. Coronavirus Antigens: Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens (Adv. Biology 4/2021)
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Sung-Ah Hong, Keun Bon Ku, Seongjun Kim, Bum-Tae Kim, Sangsu Bae, Hyung Jin Cha, Chonsaeng Kim, Jihyeon Yu, Jun Hee Han, You Kyeong Jeong, Hye Jin Shin, and Jae Sung Woo
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Biomaterials ,Antigen ,Biomedical Engineering ,medicine ,Cover Picture ,Human cell ,Biology ,medicine.disease_cause ,Virology ,General Biochemistry, Genetics and Molecular Biology ,Coronavirus - Abstract
The production of biomolecules that require human‐specific lipid environments is extremely useful for basic research and medical applications. In article number 2000154, Seong‐Jun Kim, Jae‐Sung Woo, Sangsu Bae, and co‐workers integrate multiple proteins or virus antigens into defined transcriptional hotspots in the human genome via a homology‐independent targeted insertion method using CRISPR nucleases. This system is similar to a production pipeline of biomolecules in a factory controlled by CRISPR.
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- 2021
7. Purification of Therapeutic Antibodies Using the Ca2+-Dependent Phase-Transition Properties of Calsequestrin.
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Heesun Park, Hyungsu Jeon, Hyung Jin Cha, Jinho Bang, Youngwoo Song, Mihyun Choi, Daekyung Sung, Won Il Choi, Jin Hyung Lee, Jae-Sung Woo, Sangyong Jon, and Sunghyun Kim
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- 2022
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8. Discovery of direct-acting antiviral agents with a graphene-based fluorescent nanosensor
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Dal-Hee Min, Jungho Kim, Yong Suk Jang, Hojeong Shin, Cheolhee Won, Hyung Jin Cha, Seounghun Kang, Jisang Park, Jae Sung Woo, and Se Jin Park
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Drug ,medicine.drug_class ,viruses ,media_common.quotation_subject ,Diseases and Disorders ,Dengue virus ,medicine.disease_cause ,03 medical and health sciences ,chemistry.chemical_compound ,In vivo ,RNA polymerase ,medicine ,Health and Medicine ,Research Articles ,030304 developmental biology ,media_common ,chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,030306 microbiology ,RNA ,SciAdv r-articles ,Virology ,In vitro ,Enzyme ,chemistry ,Antiviral drug ,Research Article - Abstract
We report a novel high-throughput screening tool for drug discovery of direct-acting antiviral agents against RNA viruses., Direct-acting agents against viral components are considered as the most promising candidates for the successful antiviral therapeutics. To date, no direct-acting drugs exist for the treatment against dengue virus (DV) infection, which can develop into life-threatening diseases. RNA-dependent RNA polymerase (RdRp), an RNA virus–specific enzyme highly conserved among various viral families, has been known as the broad-range antiviral drug target. Here, we developed an RNA-based graphene biosensor system [RNA nano-graphene oxide system (RANGO)] to enable the fluorescence-based quantitative analysis of the RdRp enzyme activity. We used the RANGO system to a high-throughput chemical screening to identify novel direct-acting antiviral drug candidates targeting DV RdRp from the FDA-approved small-molecule library. RANGO accelerated the massive selection of drug candidates. We found that one of the selected hit compounds, montelukast, showed antiviral activity in vitro and in vivo by directly inhibiting replication of DV and thus relieved related symptoms.
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- 2020
9. A fluorescent nanobiosensor for the facile analysis of m
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Seo-Hee, Yim, Hyung Jin, Cha, Se-Jin, Park, Yeajee, Yim, Jae-Sung, Woo, and Dal-Hee, Min
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DNA-Binding Proteins ,Spectrometry, Fluorescence ,Escherichia coli Proteins ,Endoribonucleases ,AlkB Homolog 5, RNA Demethylase ,Nanoparticles ,Graphite ,Biosensing Techniques ,Demethylation - Abstract
RNA demethylase has recently been known to be associated with cancer development but its selective inhibitors as anti-cancer agents have rarely been investigated to date. Herein, we have developed a fluorescent nanobiosensor which enables efficient quantitative analysis of RNA demethylase ALKBH5 activity and shows a high potential for robust inhibitor screening.
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- 2020
10. Structural analyses combined with small-angle X-ray scattering reveals that the retention of heme is critical for maintaining the structure of horseradish peroxidase under denaturing conditions
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Kwan Yong Choi, Hyung Jin Cha, Kyeong Sik Jin, and Do Soo Jang
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Models, Molecular ,0301 basic medicine ,Protein Denaturation ,Circular dichroism ,Clinical Biochemistry ,Heme ,macromolecular substances ,environment and public health ,Biochemistry ,Horseradish peroxidase ,03 medical and health sciences ,chemistry.chemical_compound ,X-Ray Diffraction ,Urea ,Guanidine ,Protein secondary structure ,Horseradish Peroxidase ,Protein Unfolding ,030102 biochemistry & molecular biology ,biology ,Small-angle X-ray scattering ,Circular Dichroism ,Organic Chemistry ,Temperature ,food and beverages ,Protein tertiary structure ,Protein Structure, Tertiary ,Crystallography ,030104 developmental biology ,chemistry ,biology.protein - Abstract
We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9 M urea or 6 M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9 M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9 M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9 M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6 M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9 M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.
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- 2017
11. Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens
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Bum Tae Kim, Jun Hee Han, Sangsu Bae, You Kyeong Jeong, Jihyeon Yu, Hyung Jin Cha, Sung Ah Hong, Keun Bon Ku, Chonsaeng Kim, Seongjun Kim, Jae Sung Woo, and Hye Jin Shin
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Biomedical Engineering ,Gene Expression ,Computational biology ,Biology ,Antibodies, Viral ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,Coronavirus OC43, Human ,Biomaterials ,Mice ,Antigen ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,CRISPR ,Antigens, Viral ,Vero Cells ,Gene ,Coronavirus ,virus diseases ,Human cell ,Antibodies, Neutralizing ,Human coronavirus ,Recombinant Proteins ,HEK293 Cells ,Middle East Respiratory Syndrome Coronavirus ,biology.protein ,Female ,Human genome ,Antibody - Abstract
Coproduction of multiple proteins at high levels in a single human cell line would be extremely useful for basic research and medical applications. Here, a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome is presented. As a proof-of-concept, it is shown that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number. It is confirmed that three different fluorescent proteins, encoded by genes integrated at different loci, can be coexpressed at high levels. Further, a stable cell line is generated, producing antigens from different human coronaviruses: MERS-CoV and HCoV-OC43. Antibodies raised against these antigens, which contain human N-glycosylation, show neutralizing activities against both viruses, suggesting that the coexpression system provides a quick and predictable way to produce multiple coronavirus antigens, such as the recent 2019 novel human coronavirus.
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- 2021
12. Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens.
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Chonsaeng Kim, You Kyeong Jeong, Jihyeon Yu, Hye Jin Shin, Keun Bon Ku, Hyung Jin Cha, Jun Hee Han, Sung-Ah Hong, Bum-Tae Kim, Seong-Jun Kim, Jae-Sung Woo, and Sangsu Bae
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SARS-CoV-2 ,CORONAVIRUSES ,COVID-19 ,ANTIGENS ,FLUORESCENT proteins ,HUMAN genome ,CORONAVIRUS diseases - Abstract
Coproduction of multiple proteins at high levels in a single human cell line would be extremely useful for basic research and medical applications. Here, a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome is presented. As a proof-of-concept, it is shown that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number. It is confirmed that three different fluorescent proteins, encoded by genes integrated at different loci, can be coexpressed at high levels. Further, a stable cell line is generated, producing antigens from different human coronaviruses: MERS-CoV and HCoV-OC43. Antibodies raised against these antigens, which contain human N-glycosylation, show neutralizing activities against both viruses, suggesting that the coexpression system provides a quick and predictable way to produce multiple coronavirus antigens, such as the recent 2019 novel human coronavirus. [ABSTRACT FROM AUTHOR]
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- 2021
- Full Text
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13. Crystal Structures of Penicillin-Binding Protein D2 from Listeria monocytogenes and Structural Basis for Antibiotic Specificity
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Hyung Jin Cha, Yeon-Gil Kim, and Jae-Hee Jeong
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0301 basic medicine ,Penicillin binding proteins ,Cefotaxime ,medicine.drug_class ,030106 microbiology ,Antibiotics ,Crystallography, X-Ray ,beta-Lactams ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Listeria monocytogenes ,Ampicillin ,polycyclic compounds ,medicine ,Humans ,Penicillin-Binding Proteins ,Pharmacology (medical) ,Mechanisms of Action: Physiological Effects ,Pharmacology ,Cefuroxime ,Cephem ,Chemistry ,Penicillin G ,biochemical phenomena, metabolism, and nutrition ,Anti-Bacterial Agents ,Penicillin ,030104 developmental biology ,Infectious Diseases ,Penam ,medicine.drug - Abstract
β-Lactam antibiotics that inhibit penicillin-binding proteins (PBPs) have been widely used in the treatment of bacterial infections. However, the molecular basis underlying the different inhibitory potencies of β-lactams against specific PBPs is not fully understood. Here, we present the crystal structures of penicillin-binding protein D2 (PBPD2) from Listeria monocytogenes, a Gram-positive foodborne bacterial pathogen that causes listeriosis in humans. The acylated structures in complex with four antibiotics (penicillin G, ampicillin, cefotaxime, and cefuroxime) revealed that the β-lactam core structures were recognized by a common set of residues; however, the R1 side chains of each antibiotic participate in different interactions with PBPD2. In addition, the structural complementarities between the side chains of β-lactams and the enzyme were found to be highly correlated with the relative reactivities of penam or cephem antibiotics against PBPD2. Our study provides the structural basis for the inhibition of PBPD2 by clinically important β-lactam antibiotics that are commonly used in listeriosis treatment. Our findings imply that the modification of β-lactam side chains based on structural complementarity could be useful for the development of potent inhibitors against β-lactam-resistant PBPs.
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- 2018
14. Non-Zero Grid for Accurate 2-Bit Additive Power-of-Two CNN Quantization
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Young Min Kim, Kyunghyun Han, Wai-Kong Lee, Hyung Jin Chang, and Seong Oun Hwang
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Quantization ,deep learning ,convolutional neural network ,Internet of Things ,Electrical engineering. Electronics. Nuclear engineering ,TK1-9971 - Abstract
Quantization is an effective technique to reduce the memory and computational complexity of CNNs. Recent advances utilize additive powers-of-two to perform non-uniform quantization, which resembles a normal distribution and shows better performance than uniform quantization. With powers-of-two quantization, the computational complexity is also largely reduced because the slow multiplication operations are replaced with lightweight shift operations. However, there are serious problems in the previously proposed grid formulation for 2-bit quantization. In particular, these powers-of-two schemes produce zero values, generating significant training error and causing low accuracy. In addition, due to improper grid formulation, they also fallback to uniform quantization when the quantization level reaches 2-bit. Due to these reasons, on large CNN like ResNet-110, these powers-of-two schemes may not even train properly. To resolve these issues, we propose a new non-zero grid formulation that enables 2-bit non-uniform quantization and allow the CNN to be trained successfully in every attempt, even for a large network. The proposed technique quantizes weight as power-of-two values and projects it close to the mean area through a simple constant product on the exponential part. This allows our quantization scheme to closely resemble a non-uniform quantization at 2-bit, enabling successful training at 2-bit quantization, which is not found in the previous work. The proposed technique achieves 70.57% accuracy on the CIFAR-100 dataset trained with ResNet-110. This result is 6.24% higher than the additive powers-of-two scheme which only achieves 64.33% accuracy. Beside achieving higher accuracy, our work also maintains the same memory and computational efficiency with the original additive powers-of-two scheme.
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- 2023
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15. Three-Dimensional Structures of a Wild-Type Ketosteroid Isomerase and Its Single Mutant in Solution
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Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Jehan Kim, Kyeong Sik Jin, Hee Cheon Lee, Hyeong Ju Lee, Moonhor Ree, Do Soo Jang, and Eung-Sam Kim
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chemistry.chemical_compound ,Materials science ,chemistry ,Biochemistry ,Ketosteroid ,Mutant ,Wild type ,General Materials Science ,Isomerase - Published
- 2014
16. Structure of the hypothetical protein Ton 1535 from Thermococcus onnurineus NA1 reveals unique structural properties by a left-handed helical turn in normal α-solenoid protein
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Catleya Rojvirija, Yi-Seul Kim, Yeon-Gil Kim, Hyung Jin Cha, Jae-Hee Jeong, and Sung Chul Ha
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Left handed ,Crystallography ,Thermococcus onnurineus ,Materials science ,Structural Biology ,Hypothetical protein ,Crystal structure ,Antiparallel (biochemistry) ,Molecular Biology ,Biochemistry - Abstract
The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 A resolution. With two antiparallel α-helices in a helix-turn-helix motif as a repeating unit, Ton1535 consists of right-handed coiled N- and C-terminal regions that are stacked together using helix bundles containing a left-handed helical turn. One left-handed helical turn in the right-handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α-helices on the convex surfaces of the N-terminal region to the extended surface of the concave groove of the C-terminal region and vice versa. Proteins 2014; 82:1072–1078. © 2013 Wiley Periodicals, Inc.
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- 2013
17. Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase
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Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Jae Sung Woo, Do Soo Jang, Yeon Gil Kim, and Kyong-Tai Kim
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Protein Folding ,Mutant ,Steroid Isomerases ,Isomerase ,Crystallography, X-Ray ,medicine.disease_cause ,Hydrophobic effect ,chemistry.chemical_compound ,Isomerism ,Catalytic Domain ,Ketosteroid ,Enzyme Stability ,medicine ,Urea ,Molecular Biology ,Mutation ,biology ,Pseudomonas putida ,Active site ,Hydrogen Bonding ,Articles ,Cell Biology ,General Medicine ,Kinetics ,Amino Acid Substitution ,chemistry ,Biochemistry ,Biocatalysis ,Biophysics ,biology.protein ,Mutant Proteins ,Protein folding ,Function (biology) - Abstract
Proteins have evolved to compensate for detrimental mutations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isomerase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Previous results revealed that the Y30F mutation rescued the Y14F, Y55F and Y14F/Y55F mutants by increasing the catalytic activity by 23-, 3- and 1.3-fold, respectively, and the Y55F mutant by increasing the stability by 3.3 kcal/mol. To better understand these observations, we systematically investigated detailed structural and thermodynamic effects of the Y30F mutation on these mutants. Crystal structures of the Y14F/Y30F and Y14F/Y55F mutants were solved at 2.0 and 1.8 Å resolution, respectively, and compared with previoulsy solved structures of wild-type and other mutant KSIs. Structural analyses revealed that the Y30F mutation partially restored the active-site cleft of these mutant KSIs. The Y30F mutation also increased Y14F and Y14F/Y55F mutant stability by 3.2 and 4.3 kcal/mol, respectively, and the melting temperatures of the Y14F, Y55F and Y14F/Y55F mutants by 6.4°C, 5.1°C and 10.0°C, respectively. Compensatory effects of the Y30F mutation on stability might be due to improved hydrophobic interactions because removal of a hydroxyl group from Tyr30 induced local compaction by neighboring residue movement and enhanced interactions with surrounding hydrophobic residues in the active site. Taken together, our results suggest that perturbed active-site geometry recovery and favorable hydrophobic interactions mediate the role of Y30F as a second-site suppressor.
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- 2013
18. Role of conserved Met112 residue in the catalytic activity and stability of ketosteroid isomerase
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Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Jae-Hee Jeong, Eun Ju Shin, Young Sung Yun, and Do Soo Jang
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0301 basic medicine ,Stereochemistry ,Biophysics ,Steroid Isomerases ,Isomerase ,Biochemistry ,Catalysis ,Analytical Chemistry ,Enzyme catalysis ,03 medical and health sciences ,chemistry.chemical_compound ,Methionine ,Ketosteroid ,Catalytic Domain ,Organic chemistry ,Transition Temperature ,Enzyme kinetics ,Amino Acid Sequence ,Molecular Biology ,Alanine ,030102 biochemistry & molecular biology ,biology ,Hydrogen bond ,Pseudomonas putida ,Active site ,Hydrogen Bonding ,biology.organism_classification ,Ketosteroids ,030104 developmental biology ,chemistry ,Amino Acid Substitution ,Mutation ,biology.protein ,Hydrophobic and Hydrophilic Interactions ,Sequence Alignment - Abstract
Ketosteroid isomerase (3-oxosteroid Δ(5)-Δ(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Δ(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the kcat by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased KD values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (Tm) by 6.4°C and 2.5°C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased kcat by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing Tm by 4.1°C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core.
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- 2016
19. Rapid Mapping of Active Site of KSI by Paramagnetic NMR
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Hyeong Ju Lee, Kwan Yong Choi, Hyung Jin Cha, Yong Nam Joe, and Hee Cheon Lee
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biology ,Stereochemistry ,Active site ,Substrate (chemistry) ,General Chemistry ,Crystal structure ,Isomerase ,chemistry.chemical_compound ,chemistry ,Reagent ,Ketosteroid ,medicine ,biology.protein ,Equilenin ,Heteronuclear single quantum coherence spectroscopy ,medicine.drug - Abstract
N HSQC spectra using 4-hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and an intermediateanalog (equilenin). Our result revealed that residues in hydrophobic cavity of KSI, particularly active siteregion, mainly experienced a high line-broadening effect of NMR signal with HyTEMPO, while theyexperienced full recovery of a lineshape upon the addition of equilenin. The mapped region was very similarto the active site of KSI as described by the crystal structure. These observations indicate that a combined useof paramagnetic reagent and substrate (or analog) could rapidly identify the residues in potential active site ofKSI, and can be applied to the analysis of both active site and function in unknown protein.Key Words : NMR, Ketosteroid isomerase, Active site, Paramagnetic agent, EquileninIntroductionIt has been known that the protein active site plays asignificant role in various physiological processes. Thecatalytic process is a binding reaction of protein similar to alock and key model where the substrate behaves like a keyfitting in the lock by interaction between substrate andresidues of active site in the protein. The active site of theprotein fits to the substrate conformation in size, shape,charge, and hydrophobic or hydrophilic character, or a littleconformational change is induced in order to ensure precisebinding to the substrate.
- Published
- 2012
20. Rapamycin inhibits both motility through down-regulation of p-STAT3 (S727) by disrupting the mTORC2 assembly and peritoneal dissemination in sarcomatoid cholangiocarcinoma
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Dae Ghon Kim, Jung Hee Kwon, Nam Gyu Lee, Kwan Yong Choi, Hyung Jin Cha, Sun Mi Hong, Hong Gil Nam, Chang Wook Park, and Young Sung Yun
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Male ,STAT3 Transcription Factor ,Cancer Research ,Blotting, Western ,Down-Regulation ,Mice, Nude ,Motility ,Apoptosis ,Mechanistic Target of Rapamycin Complex 2 ,Biology ,Real-Time Polymerase Chain Reaction ,mTORC2 ,Cholangiocarcinoma ,Immunoenzyme Techniques ,Mice ,Cell Movement ,Cell Adhesion ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Immunoprecipitation ,RNA, Messenger ,Phosphorylation ,Protein kinase B ,Peritoneal Neoplasms ,PI3K/AKT/mTOR pathway ,Cell Proliferation ,Sirolimus ,Reverse Transcriptase Polymerase Chain Reaction ,TOR Serine-Threonine Kinases ,RPTOR ,General Medicine ,Bile Ducts, Intrahepatic ,Bile Duct Neoplasms ,Oncology ,Multiprotein Complexes ,Cancer research ,Proto-Oncogene Proteins c-akt ,medicine.drug - Abstract
Cholangiocarcinoma (CC) is a malignant epithelium neoplasm that originates from the bile epithelium and for which there are few therapeutic strategies. The mTOR pathway involved in many cellular processes was reported to be up-regulated in various cancers. We investigated the activation of the AKT/mTOR pathway in CC cell lines with different degrees of dedifferentiation and found that rapamycin could suppress the motility and the peritoneal dissemination of sarcomatoid SCK cells. Inhibition of the mTOR pathway with rapamycin decreased significantly the number of tumor nodules and prolonged the survival rates of nude mice inoculated with sarcomatoid CC cells. Prolonged treatments with rapamycin were found to disrupt the mTORC2 assembly and to reduce the phosphorylation of STAT3 at Ser 727. Rapamycin decreased both mRNA and protein levels of MMP2 and Twist1, which are regulated by STAT3 and associated with cancer metastasis. The overexpression of STAT3 S727A lacking the phosphorylation site resulted in significantly less sensitivity to rapamycin than the overexpression of STAT3 WT. Taken together, our results suggest that rapamycin could suppress the motility of sarcomatoid CC by down-regulating p-STAT3 (S727) through the impairment of mTORC2 assembly.
- Published
- 2012
21. The expression of phospho-AKT1 and phospho-MTOR is associated with a favorable prognosis independent of PTEN expression in intrahepatic cholangiocarcinomas
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Kwan Yong Choi, Hyung Jin Cha, Chang Ohk Sung, D.W. Choi, Dae Shick Kim, Dakeun Lee, Jin Young Park, Jongmin Kim, Jin Seok Heo, Jae-Won Joh, Kyusam Choi, Kee-Taek Jang, Seoung Ho Choi, and In-Gu Do
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Adult ,Male ,Pathology ,medicine.medical_specialty ,Adolescent ,Blotting, Western ,AKT1 ,Cell Cycle Proteins ,Kaplan-Meier Estimate ,Biology ,medicine.disease_cause ,Pathology and Forensic Medicine ,Cholangiocarcinoma ,Young Adult ,Republic of Korea ,Biomarkers, Tumor ,medicine ,Humans ,Tensin ,PTEN ,Phosphorylation ,PI3K/AKT/mTOR pathway ,Intrahepatic Cholangiocarcinoma ,Adaptor Proteins, Signal Transducing ,Aged ,Proportional Hazards Models ,Chi-Square Distribution ,TOR Serine-Threonine Kinases ,PTEN Phosphohydrolase ,Ribosomal Protein S6 Kinases, 70-kDa ,Middle Aged ,Phosphoproteins ,Prognosis ,Immunohistochemistry ,Molecular biology ,Up-Regulation ,Bile Ducts, Intrahepatic ,Bile Duct Neoplasms ,Ribosomal protein s6 ,embryonic structures ,biology.protein ,Cancer research ,Female ,Carcinogenesis ,Proto-Oncogene Proteins c-akt - Abstract
AKT1 signaling pathway is important for the regulation of protein synthesis and cell survival with implications in carcinogenesis. In this study, we explored the prognostic significance of AKT1 pathway in intrahepatic cholangiocarcinomas. We investigated the status of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), phosphorylated (p) AKT1 (p-AKT1), p-mammalian target of rapamycin (p-MTOR), p-p70 ribosomal protein S6 kinase (p-RPS6KB2) and p-eukaryotic initiation factor 4E-binding protein-1 (p-EIF4EBP1) in 101 intrahepatic cholangiocarcinomas by immunohistochemistry. Western blot analysis was performed to verify the expression levels of p-AKT1 and p-MTOR. The relationship of protein expression with clinicopathological data and the correlations of protein expression levels were explored. The overexpression of p-AKT1, p-MTOR, and PTEN was associated with a better survival in patients with intrahepatic cholangiocarcinoma (P=0.0137, 0.0194, and 0.0337, respectively). In a multivariate analysis, PTEN was an independent prognostic factor, and p-AKT1 showed tendency (P=0.032 and 0.051, respectively). The overexpression of p-MTOR was correlated with well-to-moderately differentiated tumors (P
- Published
- 2012
22. Structure of Putrescine Aminotransferase from Escherichia coli Provides Insights into the Substrate Specificity among Class III Aminotransferases
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Yeon-Gil Kim, Hyung Jin Cha, Jae-Hee Jeong, and Catleya Rojviriya
- Subjects
Protein Denaturation ,Protein Conformation ,Enzyme Metabolism ,Molecular Sequence Data ,lcsh:Medicine ,Research and Analysis Methods ,Crystallography, X-Ray ,Biochemistry ,Microbiology ,Cofactor ,Substrate Specificity ,chemistry.chemical_compound ,Protein structure ,Escherichia coli ,Putrescine ,Transferase ,Amino Acid Sequence ,Binding site ,Pyridoxal phosphate ,lcsh:Science ,Enzyme Chemistry ,Peptide sequence ,Transaminases ,chemistry.chemical_classification ,Multidisciplinary ,biology ,Sequence Homology, Amino Acid ,lcsh:R ,Biology and Life Sciences ,Bacteriology ,Macromolecular Crystallography ,Bacterial Biochemistry ,Enzyme ,chemistry ,Pyridoxal Phosphate ,biology.protein ,Enzymology ,Crystallographic Techniques ,Cofactors (Biochemistry) ,lcsh:Q ,Dimerization ,Research Article - Abstract
YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 angstrom resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.
- Published
- 2014
23. Contribution of a low-barrier hydrogen bond to catalysis is not significant in ketosteroid isomerase
- Author
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Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Sejeong Shin, Do Soo Jang, Gildon Choi, Hyeong Ju Lee, and Hee Cheon Lee
- Subjects
Models, Molecular ,low-barrier hydrogen bond ,Stereochemistry ,Proton Magnetic Resonance Spectroscopy ,Low-barrier hydrogen bond ,Steroid Isomerases ,Isomerase ,Reaction intermediate ,Crystallography, X-Ray ,enzyme catalysis ,Article ,Catalysis ,Enzyme catalysis ,Substrate Specificity ,chemistry.chemical_compound ,Bacterial Proteins ,Ketosteroid ,Catalytic Domain ,Molecular Biology ,Equilenin ,Tyr14 ,biology ,Hydrogen bond ,Pseudomonas putida ,Active site ,Hydrogen Bonding ,Cell Biology ,General Medicine ,chemistry ,Biochemistry ,Mutation ,biology.protein ,Biocatalysis ,ketosteroid isomerase ,Protein Binding - Abstract
Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ(5)-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ(5)-3-ketosteroid to its conjugated Δ(4)-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1-11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7-2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.
- Published
- 2014
24. Structural insights into the histidine trimethylation activity of EgtD from Mycobacterium smegmatis
- Author
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Hyung Jin Cha, Jae-Hee Jeong, Yeon-Gil Kim, Catleya Rojviriya, and Sung-Chul Ha
- Subjects
Models, Molecular ,Conformational change ,Methyltransferase ,Stereochemistry ,Protein Conformation ,Mycobacterium smegmatis ,Biophysics ,Biochemistry ,Methylation ,chemistry.chemical_compound ,Ergothioneine biosynthetic process ,Imidazole ,Transferase ,Histidine ,Protein Methyltransferases ,Molecular Biology ,Binding Sites ,biology ,Cell Biology ,biology.organism_classification ,Protein Structure, Tertiary ,Betaine ,chemistry ,Models, Chemical ,Ergothioneine ,Protein Binding - Abstract
EgtD is an S-adenosyl-l-methionine (SAM)-dependent histidine N,N,N-methyltransferase that catalyzes the formation of hercynine from histidine in the ergothioneine biosynthetic process of Mycobacterium smegmatis. Ergothioneine is a secreted antioxidant that protects mycobacterium from oxidative stress. Here, we present three crystal structures of EgtD in the apo form, the histidine-bound form, and the S-adenosyl-l-homocysteine (SAH)/histidine-bound form. The study revealed that EgtD consists of two distinct domains: a typical methyltransferase domain and a unique substrate binding domain. The histidine binding pocket of the substrate binding domain primarily recognizes the imidazole ring and carboxylate group of histidine rather than the amino group, explaining the high selectivity for histidine and/or (mono-, di-) methylated histidine as substrates. In addition, SAM binding to the MTase domain induced a conformational change in EgtD to facilitate the methyl transfer reaction. The structural analysis provides insights into the putative catalytic mechanism of EgtD in a processive trimethylation reaction.
- Published
- 2014
25. Crystallization and preliminary X-ray crystallographic analysis of PBPD2 from Listeria monocytogenes
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Jae-Hee Jeong, Hyung Jin Cha, and Yeon-Gil Kim
- Subjects
Penicillin binding proteins ,Biophysics ,Biology ,medicine.disease_cause ,Crystallography, X-Ray ,Biochemistry ,Bacterial cell structure ,law.invention ,chemistry.chemical_compound ,Listeria monocytogenes ,Structural Biology ,law ,Genetics ,medicine ,Escherichia coli ,Penicillin-Binding Proteins ,Crystallization ,Resolution (electron density) ,Condensed Matter Physics ,Crystallography ,chemistry ,Crystallization Communications ,Orthorhombic crystal system ,Peptidoglycan - Abstract
Penicillin-binding proteins (PBPs), which mediate the peptidoglycan biosynthetic pathway in the bacterial cell wall, have been intensively investigated as a target for the design of antibiotics. In this study, PBPD2, a low-molecular-weight PBP encoded bylmo2812fromListeria monocytogenes, was overexpressed inEscherichia coli, purified and crystallized at 295 K using the sitting-drop vapour-diffusion method. The crystal belonged to the primitive orthorhombic space groupP212121, with unit-cell parametersa= 37.7,b= 74.7,c= 75.1 Å, and diffracted to 1.55 Å resolution. There was one molecule in the asymmetric unit. The preliminary structure was determined by the molecular-replacement method.
- Published
- 2013
26. Structure of the hypothetical protein Ton1535 from Thermococcus onnurineus NA1 reveals unique structural properties by a left-handed helical turn in normal α-solenoid protein
- Author
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Jae-Hee, Jeong, Yi-Seul, Kim, Catleya, Rojvirija, Hyung Jin, Cha, Yeon-Gil, Kim, and Sung Chul, Ha
- Subjects
Models, Molecular ,Thermococcus ,Archaeal Proteins ,Molecular Sequence Data ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Crystallography, X-Ray ,Hydrophobic and Hydrophilic Interactions ,Protein Structure, Secondary - Abstract
The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 Å resolution. With two antiparallel α-helices in a helix-turn-helix motif as a repeating unit, Ton1535 consists of right-handed coiled N- and C-terminal regions that are stacked together using helix bundles containing a left-handed helical turn. One left-handed helical turn in the right-handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α-helices on the convex surfaces of the N-terminal region to the extended surface of the concave groove of the C-terminal region and vice versa.
- Published
- 2013
27. The structure of TON1937 from archaeon Thermococcus onnurineus NA1 reveals a eukaryotic HEAT-like architecture
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Hyung Jin Cha, Jae-Hee Jeong, Catleya Rojviriya, Yeon-Gil Kim, Yi-Seul Kim, and Sung-Chul Ha
- Subjects
Models, Molecular ,Structural similarity ,Protein Conformation ,Archaeal Proteins ,Hypothetical protein ,Molecular Sequence Data ,Sequence alignment ,Ligands ,Biochemistry ,Structural Biology ,Molecular evolution ,Consensus sequence ,Amino Acid Sequence ,Molecular Biology ,Genetics ,Armadillo Domain Proteins ,Thermococcus onnurineus ,Binding Sites ,biology ,General Medicine ,Protein superfamily ,biology.organism_classification ,Recombinant Proteins ,Thermococcus ,Sequence Alignment ,Archaea ,Protein Binding - Abstract
The members of the ARM/HEAT repeat-containing protein superfamily in eukaryotes have been known to mediate protein-protein interactions by using their concave surface. However, little is known about the ARM/HEAT repeat proteins in prokaryotes. Here we report the crystal structure of TON1937, a hypothetical protein from the hyperthermophilic archaeon Thermococcus onnurineus NA1. The structure reveals a crescent-shaped molecule composed of a double layer of α-helices with seven anti-parallel α-helical repeats. A structure-based sequence alignment of the α-helical repeats identified a conserved pattern of hydrophobic or aliphatic residues reminiscent of the consensus sequence of eukaryotic HEAT repeats. The individual repeats of TON1937 also share high structural similarity with the canonical eukaryotic HEAT repeats. In addition, the concave surface of TON1937 is proposed to be its potential binding interface based on this structural comparison and its surface properties. These observations lead us to speculate that the archaeal HEAT-like repeats of TON1937 have evolved to engage in protein-protein interactions in the same manner as eukaryotic HEAT repeats.
- Published
- 2013
28. Controlled release of human growth hormone fused with a human hybrid Fc fragment through a nanoporous polymer membrane
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Kyeong Sik Jin, Mi Nam Lee, Do Soo Jang, Jin Kon Kim, Eung-Sam Kim, Young Chul Sung, Seung Yun Yang, Kwan Yong Choi, and Hyung Jin Cha
- Subjects
endocrine system ,Circular dichroism ,Materials science ,Recombinant Fusion Proteins ,law.invention ,Cell Line ,law ,Humans ,General Materials Science ,Phosphorylation ,B-Lymphocytes ,Nanoporous ,Human Growth Hormone ,Protein Stability ,Membranes, Artificial ,Immunoglobulin D ,Janus Kinase 2 ,Controlled release ,Fusion protein ,Immunoglobulin Fc Fragments ,Membrane ,Biochemistry ,Delayed-Action Preparations ,Immunoglobulin G ,Drug delivery ,Recombinant DNA ,Biophysics ,Porosity ,hormones, hormone substitutes, and hormone antagonists - Abstract
Nanotechnology has been applied to the development of more effective and compatible drug delivery systems for therapeutic proteins. Human growth hormone (hGH) was fused with a hybrid Fc fragment containing partial Fc domains of human IgD and IgG4 to produce a long-acting fusion protein. The fusion protein, hGH-hyFc, resulted in the increase of the hydrodynamic diameter (ca. 11 nm) compared with the diameter (ca. 5 nm) of the recombinant hGH. A diblock copolymer membrane with nanopores (average diameter of 14.3 nm) exhibited a constant release rate of hGH-hyFc. The hGH-hyFc protein released in a controlled manner for one month was found to trigger the phosphorylation of Janus kinase 2 (JAK2) in human B lymphocyte and to exhibit an almost identical circular dichroism spectrum to that of the original hGH-hyFc, suggesting that the released fusion protein should maintain the functional and structural integrity of hGH. Thus, the nanoporous release device could be a potential delivery system for the long-term controlled release of therapeutic proteins fused with the hybrid Fc fragment.
- Published
- 2013
29. Apoptosis-related mRNA expression profiles of ovarian cancer cell lines following cisplatin treatment
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Sung Jong Lee, Bee Hak Hong, Joo-Hee Yoon, Kwan Yong Choi, Hyung Jin Cha, Chang Wook Park, and Eung-Sam Kim
- Subjects
Cisplatin ,Programmed cell death ,endocrine system diseases ,DNA repair ,Obstetrics and Gynecology ,General Medicine ,Cell sorting ,Biology ,Bioinformatics ,medicine.disease ,female genital diseases and pregnancy complications ,Oncology ,Apoptosis ,Cell culture ,medicine ,Cancer research ,Original Article ,Ovarian cancer ,Gene ,medicine.drug - Abstract
Objective The aim of this study was to identify apoptosis-related genes of ovarian cancer cell lines following cisplatin treatment. Methods We used IC(50) values and fluorescence-activated cell sorting analysis to compare cell death in 2 ovarian cancer cell lines, namely, SKOV-3 and OVCAR-3, upon treatment with cisplatin. Moreover, the change in transcriptional levels of apoptosis-associated genes was measured with a dendron-modified DNA microarray. Results The protein levels for the up-regulated genes in each cell line were validated to identify the molecules that may determine the cellular behavior of cisplatin resistance. Eight genes were over-expressed in the 2 cell lines. The cisplatin-induced up-regulation of DAD1 in transcriptional and protein levels contributed to the cisplatin resistance of OVCAR-3, and the up-regulation of FASTK and TNFRSF11A in SKOV-3 resulted in its higher sensitivity to cisplatin than that of OVCAR-3. Conclusion In the present study, we have identified a set of genes responsible for apoptosis following cisplatin treatment in ovarian cancer cell lines. These genes may give information about the understanding of cisplatin-induced apoptosis in ovarian cancer.
- Published
- 2010
30. Self-Supervised Visual Learning by Variable Playback Speeds Prediction of a Video
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Hyeon Cho, Taehoon Kim, Hyung Jin Chang, and Wonjun Hwang
- Subjects
Action recognition ,representation learning ,self-supervised learning ,Electrical engineering. Electronics. Nuclear engineering ,TK1-9971 - Abstract
We propose a self-supervised visual learning method by predicting the variable playback speeds of a video. Without semantic labels, we learn the spatio-temporal visual representation of the video by leveraging the variations in the visual appearance according to different playback speeds under the assumption of temporal coherence. To learn the spatio-temporal visual variations in the entire video, we have not only predicted a single playback speed but also generated clips of various playback speeds and directions with randomized starting points. Hence the visual representation can be successfully learned from the meta information (playback speeds and directions) of the video. We also propose a new layer-dependable temporal group normalization method that can be applied to 3D convolutional networks to improve the representation learning performance where we divide the temporal features into several groups and normalize each one using the different corresponding parameters. We validate the effectiveness of our method by fine-tuning it to the action recognition and video retrieval tasks on UCF-101 and HMDB-51. All the source code is released in https://github.com/hyeon-jo/PSPNet.
- Published
- 2021
- Full Text
- View/download PDF
31. Structural insights of the nucleotide-dependent conformational changes of Thermotoga maritima MutL using small-angle X-ray scattering analysis
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Kwan Yong Choi, Hyung Jin Cha, Hyung Ju Lee, Tae Gyun Kim, Changill Ban, Seong-Dal Heo, and Ja Kang Ku
- Subjects
Models, Molecular ,Protein Conformation ,Size-exclusion chromatography ,Molecular Sequence Data ,DNA, Single-Stranded ,medicine.disease_cause ,Biochemistry ,DNA Mismatch Repair ,Bacterial Proteins ,medicine ,Molecule ,Nucleotide ,Thermotoga maritima ,Amino Acid Sequence ,Molecular Biology ,Escherichia coli ,chemistry.chemical_classification ,Adenosine Triphosphatases ,Binding Sites ,biology ,Small-angle X-ray scattering ,Nucleotides ,Escherichia coli Proteins ,fungi ,General Medicine ,Mismatch Repair Protein ,biology.organism_classification ,Crystallography ,MutL Proteins ,chemistry ,Chromatography, Gel ,DNA mismatch repair ,Dimerization ,Sequence Alignment - Abstract
MutL is required to assist the mismatch repair protein MutS during initiation of the methyl-directed mismatch repair (MMR) response in various organisms ranging from prokaryotes to eukaryotes. Despite this necessity, the inherent propensity of MutL to aggregate has led to significant difficulties in determining its biological relationship with other MMR-related proteins. Here, we perform analysis on the thermostable MutL protein found in Thermotoga maritima MSB8 (TmL). Size exclusion chromatographic analysis indicates the lack of aggregated forms with the exception of a dimeric TmL. Small-angle X-ray scattering (SAXS) analysis reveals that the solution structures of the full-length TmL and its corresponding complexes with nucleotides and ssDNA undergo conformational changes. The elucidated TmL SAXS model is superimposed to the crystal structure of the C-terminal domain of Escherichia coli MutL. In addition, the N-terminal SAXS model of TmL exists as monomeric form, indicating that TmL has a structurally flexible N-terminal domain. TmL SAXS analysis can suggest a considerable possibility on a new 3D view of the previously unresolved full-length MutL molecule.
- Published
- 2008
32. NMR studies on the equilibrium unfolding of ketosteroid isomerase by urea
- Author
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Do Soo Jang, Hye Seon Moon, Hee Cheon Lee, Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, and Hyeong Ju Lee
- Subjects
Models, Molecular ,Protein Denaturation ,Protein Folding ,biology ,Equilibrium unfolding ,Steroid Isomerases ,General Medicine ,Isomerase ,biology.organism_classification ,Biochemistry ,Pseudomonas putida ,Crystallography ,chemistry.chemical_compound ,chemistry ,Ketosteroid ,Urea ,Organic chemistry ,Protein folding ,Molecular Biology ,Protein secondary structure ,Dimerization ,Nuclear Magnetic Resonance, Biomolecular ,Heteronuclear single quantum coherence spectroscopy - Abstract
Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of 1H-(15)N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the beta5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the beta6-strand. The results thus suggest that the interface region primarily around the beta5- and beta6-strands could play an important role as the starting positions in the unfolding process of KSI.
- Published
- 2008
33. 15N NMR relaxation studies of Y14F mutant of ketosteroid isomerase: the influence of mutation on backbone mobility
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Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Do Soo Jang, Chul Hoon Kim, Ye Jeong Yoon, Hee Cheon Lee, and Hyeong Ju Lee
- Subjects
Models, Molecular ,Stereochemistry ,Mutant ,Steroid Isomerases ,Isomerase ,Biochemistry ,chemistry.chemical_compound ,Bacterial Proteins ,Ketosteroid ,Nandrolone ,Comamonas testosteroni ,Binding site ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Binding Sites ,biology ,Nitrogen Isotopes ,Chemistry ,Hydrogen bond ,Active site ,Hydrogen Bonding ,General Medicine ,biology.organism_classification ,Enzyme ,Amino Acid Substitution ,Mutation ,biology.protein ,Tyrosine - Abstract
The backbone dynamics of Y14F mutant of Delta(5)-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by 15N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S2) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.
- Published
- 2008
34. Alteration of the substrate specificity of ketosteroid isomerase from Pseudomonas putida Biotype B
- Author
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Kwan Yong Choi, Hyung Jin Cha, Do Soo Jang, and Han Seop Shin
- Subjects
biology ,Isomerase ,biology.organism_classification ,Biochemistry ,Pseudomonas putida ,Microbiology ,chemistry.chemical_compound ,chemistry ,Ketosteroid ,Genetics ,Substrate specificity ,Molecular Biology ,Biotechnology - Published
- 2008
35. Probing the equilibrium unfolding of ketosteroid isomerase through xenon-perturbed 1H-15N multidimensional NMR spectroscopy
- Author
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Do Soo Jang, Hyeong Ju Lee, Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Hye Seon Moon, and Hee Cheon Lee
- Subjects
Models, Molecular ,Protein Denaturation ,Protein Folding ,Magnetic Resonance Spectroscopy ,Xenon ,Chemistry ,Protein Conformation ,Pseudomonas putida ,Dimer ,Equilibrium unfolding ,Steroid Isomerases ,Nuclear magnetic resonance spectroscopy ,Isomerase ,Biochemistry ,Dissociation (chemistry) ,chemistry.chemical_compound ,Crystallography ,Kinetics ,Protein structure ,Ketosteroid ,Urea ,Protein folding ,Spectroscopy - Abstract
We used xenon-perturbed 1H-15N multidimensional NMR to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Three limited regions located on the beta3-, beta5- and beta6-strands of dimeric interface were significantly perturbed by urea in the early stage of KSI unfolding, which could lead to dissociation of the dimer into structured monomers at higher denaturant concentration as the interactions in these regions are weakened. The results indicate that the use of xenon as an indirect probe for multidimensional NMR can be a useful method for the equilibrium unfolding study of protein at residue level.
- Published
- 2007
36. A triple mutant (Y14F/Y30F/Y55F) of ketosteroid isomerase is more stable than its wild type through hydrophobic interaction as well as aromatic‐aromatic interaction
- Author
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Kwan Yong Choi, Hyung Jin Cha, Do Soo Jang, and Han Seop Shin
- Subjects
Hydrophobic effect ,chemistry.chemical_compound ,Biochemistry ,Chemistry ,Ketosteroid ,Genetics ,Wild type ,Isomerase ,Molecular Biology ,Biotechnology ,Triple mutant - Published
- 2007
37. Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase
- Author
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Hee Cheon Lee, Jehan Kim, Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Ye Jeong Yoon, Kwanseop Lim, Byeongdu Lee, Hyeong Ju Lee, Moonhor Ree, Jinhwan Yoon, and Do Soo Jang
- Subjects
Circular dichroism ,Protein Denaturation ,Protein Folding ,Equilibrium unfolding ,Biophysics ,Small angle X-ray scattering ,Steroid Isomerases ,Biochemistry ,chemistry.chemical_compound ,Structural Biology ,Ketosteroid ,Genetics ,Scattering, Radiation ,Urea ,Spectroscopy ,Molecular Biology ,Pulse field gradient NMR ,Small-angle X-ray scattering ,Pseudomonas putida ,Circular Dichroism ,Cell Biology ,Size measurement ,Crystallography ,chemistry ,Proton NMR ,Compact intermediate ,Protein folding ,Dimerization ,Ketosteroid isomerase - Abstract
Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7Å in 0M urea, 17.3Å in 5.2M urea, and 25.1Å in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using 1H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.
- Published
- 2006
38. Structural double-mutant cycle analysis of a hydrogen bond network in ketosteroid isomerase from Pseudomonas putida biotype B
- Author
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Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Heung-Soo Lee, Nam-Chul Ha, Do Soo Jang, Ja Young Lee, Byung-Ha Oh, and Sun Shin Cha
- Subjects
Models, Molecular ,Protein Denaturation ,Stereochemistry ,Mutant ,Steroid Isomerases ,Isomerase ,Biochemistry ,Catalysis ,Mutant protein ,Molecular Biology ,Binding Sites ,biology ,Hydrogen bond ,Chemistry ,Pseudomonas putida ,Active site ,Hydrogen Bonding ,Cell Biology ,biology.organism_classification ,Recombinant Proteins ,Kinetics ,Mutation ,biology.protein ,Thermodynamics ,Crystallization ,Isomerization ,Research Article - Abstract
KSI (ketosteroid isomerase) catalyses an allylic isomerization reaction at a diffusion-controlled rate. A hydrogen bond network, Asp(99).Water(504).Tyr(14).Tyr(55).Tyr(30), connects two critical catalytic residues, Tyr(14) and Asp(99), with Tyr(30), Tyr(55) and a water molecule in the highly apolar active site of the Pseudomonas putida KSI. In order to characterize the interactions among these amino acids in the hydrogen bond network of KSI, double-mutant cycle analysis was performed, and the crystal structure of each mutant protein within the cycle was determined respectively to interpret the coupling energy. The DeltaDeltaG(o) values of Y14F/D99L (Tyr(14)-->Phe/Asp(99)-->Leu) KSI, 25.5 kJ/mol for catalysis and 28.9 kJ/mol for stability, were smaller than the sums (i.e. 29.7 kJ/mol for catalysis and 34.3 kJ/mol for stability) for single mutant KSIs respectively, indicating that the effect of the Y14F/D99L mutation was partially additive for both catalysis and stability. The partially additive effect of the Y14F/D99L mutation suggests that Tyr(14) and Asp(99) should interact positively for the stabilization of the transition state during the catalysis. The crystal structure of Y14F/D99L KSI indicated that the Y14F/D99L mutation increased the hydrophobic interaction while disrupting the hydrogen bond network. The DeltaDeltaG(o) values of both Y30F/D99L and Y55F/D99L KSIs for the catalysis and stability were larger than the sum of single mutants, suggesting that either Tyr(30) and Asp(99) or Tyr(55) and Asp(99) should interact negatively for the catalysis and stability. These synergistic effects of both Y30F/D99L and Y55F/D99L mutations resulted from the disruption of the hydrogen bond network. The synergistic effect of the Y55F/D99L mutation was larger than that of the Y30F/D99L mutation, since the former mutation impaired the proper positioning of a critical catalytic residue, Tyr(14), involved in the catalysis of KSI. The present study can provide insight into interpreting the coupling energy measured by double-mutant cycle analysis based on the crystal structures of the wild-type and mutant proteins.
- Published
- 2004
39. User Modelling Using Multimodal Information for Personalised Dressing Assistance
- Author
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Yixing Gao, Hyung Jin Chang, and Yiannis Demiris
- Subjects
Multimodal user modelling ,assistive dressing ,vision and force fusion ,human-robot interaction ,Electrical engineering. Electronics. Nuclear engineering ,TK1-9971 - Abstract
Assistive robots in home environments are steadily increasing in popularity. Due to significant variabilities in human behaviour, as well as physical characteristics and individual preferences, personalising assistance poses a challenging problem. In this paper, we focus on an assistive dressing task that involves physical contact with a human's upper body, in which the goal is to improve the comfort level of the individual. Two aspects are considered to be significant in improving a user's comfort level: having more natural postures and exerting less effort. However, a dressing path that fulfils these two criteria may not be found at one time. Therefore, we propose a user modelling method that combines vision and force data to enable the robot to search for an optimised dressing path for each user and improve as the human-robot interaction progresses. We compare the proposed method against two single-modality state-of-the-art user modelling methods designed for personalised assistive dressing by user studies (31 subjects). Experimental results show that the proposed method provides personalised assistance that results in more natural postures and less effort for human users.
- Published
- 2020
- Full Text
- View/download PDF
40. Erratum to: Overexpression of Renal Tumor Antigen Is Associated with Tumor Invasion and Poor Prognosis of Hepatocellular Carcinoma
- Author
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Hyung Jin Cha, Jongmin Kim, Sun Mi Hong, Seok Joo Hong, Jun Ho Park, Eung-Sam Kim, Hee-Jung Wang, Yoon Jung Choi, In-Gu Do, Jae Won Joh, Dae Shick Kim, and Kwan Yong Choi
- Subjects
Oncology ,Surgery - Published
- 2011
41. Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase.
- Author
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Do Soo Jang, Gildon Choi, Hyung Jin Cha, Sejeong Shin, Bee Hak Hong, Hyeong Ju Lee, Hee Cheon Lee, and Kwan Yong Choi
- Abstract
Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ
5 -3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ5 -3-ketosteroid to its conjugated Δ4 -isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1-11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7-2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI. [ABSTRACT FROM AUTHOR]- Published
- 2015
- Full Text
- View/download PDF
42. Apoptosis-related mRNA expression profiles of ovarian cancer cell lines following cisplatin treatment.
- Author
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JooHee Yoon, Eung-Sam Kim, Sung Jong Lee, Chang-Wook Park, Hyung Jin Cha, Bee Hak Hong, and Kwan Yong Choi
- Published
- 2010
- Full Text
- View/download PDF
43. Structural Insights of the Nucleotide-Dependent Conformational Changes of Thermotoga maritima MutL Using Small-Angle X-ray Scattering Analysis.
- Author
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Tae Gyun Kim, Hyung Jin Cha, Hyung Ju Lee, Seong-Dal Heo, Kwan Yong Choi, Ja Kang Ku, and Changill Ban
- Subjects
- *
NUCLEOTIDES , *CONFORMATIONAL analysis , *ESCHERICHIA coli , *METHYL ether , *GENETIC mutation , *X-ray scattering - Abstract
MutL is required to assist the mismatch repair protein MutS during initiation of the methyl-directed mismatch repair (MMR) response in various organisms ranging from prokaryotes to eukaryotes. Despite this necessity, the inherent propensity of MutL to aggregate has led to significant difficulties in determining its biological relationship with other MMR-related proteins. Here, we perform analysis on the thermostable MutL protein found in Thermotoga maritima MSB8 (TmL). Size exclusion chromatographic analysis indicates the lack of aggregated forms with the exception of a dimeric TmL. Small-angle X-ray scattering (SAXS) analysis reveals that the solution structures of the full-length TmL and its corresponding complexes with nucleotides and ssDNA undergo conformational changes. The elucidated TmL SAXS model is superimposed to the crystal structure of the C-terminal domain of Escherichia coli MutL. In addition, the N-terminal SAXS model of TmL exists as monomeric form, indicating that TmL has a structurally flexible N-terminal domain. TmL SAXS analysis can suggest a considerable possibility on a new 3D view of the previously unresolved full-length MutL molecule. [ABSTRACT FROM PUBLISHER]
- Published
- 2009
- Full Text
- View/download PDF
44. 15N NMR Relaxation Studies of Y14F Mutant of Ketosteroid Isomerase: The Influence of Mutation on Backbone Mobility.
- Author
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Hyeong Ju Lee, Ye Jeong Yoon, Do Soo Jang, Chul Kim, Hyung Jin Cha, Bee Hak Hong, Kwan Yong Choi, and Hee Cheon Lee
- Subjects
ISOMERASES ,BINDING sites ,SPINE ,GENETIC mutation ,BIOCHEMISTRY - Abstract
The backbone dynamics of Y14F mutant of Δ5-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by 15N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S2) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS. [ABSTRACT FROM PUBLISHER]
- Published
- 2008
- Full Text
- View/download PDF
45. NMR Studies on the Equilibrium Unfolding of Ketosteroid Isomerase by Urea.
- Author
-
Hyeong Ju Lee, Do Soo Jang, Hyung Jin Cha, Hye Seon Moon, Bee Hak Hong, Kwan Yong Choi, and Hee Cheon Lee
- Subjects
UREA ,METABOLISM ,ISOMERASES ,SPECTRUM analysis ,PSEUDOMONADACEAE ,NUCLEAR magnetic resonance - Abstract
Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of 1H-15N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the β5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the β6-strand. The results thus suggest that the interface region primarily around the β5- and β6-strands could play an important role as the starting positions in the unfolding process of KSI. [ABSTRACT FROM PUBLISHER]
- Published
- 2008
- Full Text
- View/download PDF
46. A triple mutant (YI4F/Y30F/Y55F) of ketosteroid isomerase is more stable than its wild type through hydrophobic interaction as well as aromatic-aromatic interaction.
- Author
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Hyung Jin Cha, Do Soo Jang, Han Seop Shin, and Kwan Yong Choi
- Subjects
- *
ISOMERASES , *AROMATICITY , *STEROIDS , *KETONES , *CHEMICAL bonds , *GENETIC mutation - Abstract
One of important issues in protein science is to increase the protein stability. Utilizing a ketosteroid isomerase (KSI) as a model system, we constructed a mutant KSI which has a higher stability than its WT. We focused on the role of aromatic moieties of three tyrosine residues, Tyr14, Tyr55, and Tyr30, which constitute a hydrogen bond network in KSI. Therefore, we made a series of mutants which could substitute tyrosine with phenylalanine to remove the hydroxyl group in the tyrosine residue. We measured the free-energy change for unfolding using urea as a denaturant. We investigated the interaction among these aromatic residues for stability using the double mutant cycle analysis. We found that additional mutations on Y14F, Y55F, and Y14F/Y55F, respectively, by replacement of tyrosine at the position 30 with phenylalanine increased the stability ca. 4.0 kcal/mol. Especially the triple mutant KSI (Y14F/Y30F/Y55F) was more stable 2 kcal/mol than wild type KSI. Our structural analyses suggests that the enhanced stability of the triple mutant should be due to increased both hydrophobic interaction and aromatic-aromatic interaction upon the mutation [ABSTRACT FROM AUTHOR]
- Published
- 2007
47. Discovery of direct-acting antiviral agents with a graphene-based fluorescent nanosensor.
- Author
-
Se-Jin Park, Jungho Kim, Seounghun Kang, Hyung Jin Cha, Hojeong Shin, Jisang Park, Yong-Suk Jang, Jae-Sung Woo, Cheolhee Won, and Dal-Hee Min
- Subjects
- *
ANTIVIRAL agents , *INTERFERON receptors , *LIFE sciences , *RNA replicase , *RIBAVIRIN , *MATERIALS science , *DRUG side effects , *VIRAL nonstructural proteins - Abstract
The article informs about research on discovery of direct-acting antiviral agents with a graphene-based fluorescent nanosensor. Topics discussed include direct-acting drugs exist for the treatment against dengue virus (DV) infection, which can develop into life-threatening diseases; and development of an RNA-based graphene biosensor system [RNA nano-graphene oxide system (RANGO)] to enable the fluorescence-based quantitative analysis of the RdRp enzyme activity.
- Published
- 2020
- Full Text
- View/download PDF
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