Publications by authors named "Woo-Cheol Lee"

71 Publications

Structural basis of the complementary activity of two ketosynthases in aryl polyene biosynthesis.

Sci Rep 2021 Aug 11;11(1):16340. Epub 2021 Aug 11.

Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, South Korea.

Aryl polyenes (APE) are one of the most widespread secondary metabolites among gram-negative bacteria. In Acinetobacter baumannii, strains belonging to the virulent global clone 2 (GC2) mostly contain APE biosynthesis genes; its relevance in elevated pathogenicity is of great interest. APE biosynthesis gene clusters harbor two ketosynthases (KSs): the heterodimeric KS-chain length factor complex, ApeO-ApeC, and the homodimeric ketoacyl-acyl carrier protein synthase I (FabB)-like KS, ApeR. The role of the two KSs in APE biosynthesis is unclear. We determined the crystal structures of the two KSs from a pathogenic A. baumannii strain. ApeO-ApeC and ApeR have similar cavity volumes; however, ApeR has a narrow cavity near the entrance. In vitro assay based on the absorption characteristics of polyene species indicated the generation of fully elongated polyene with only ApeO-ApeC, probably because of the funnel shaped active site cavity. However, adding ApeR to the reaction increases the throughput of APE biosynthesis. Mutagenesis at Tyr135 in the active site cavity of ApeR reduces the activity significantly, which suggests that the stacking of the aryl group between Tyr135 and Phe202 is important for substrate recognition. Therefore, the two KSs function complementarily in the generation of APE to enhance its production.
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http://dx.doi.org/10.1038/s41598-021-95890-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358021PMC
August 2021

Structural comparison of Acinetobacter baumannii β-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis.

Sci Rep 2021 Apr 12;11(1):7945. Epub 2021 Apr 12.

Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea.

Some Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the β-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the β-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure-function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host-pathogen interaction mechanisms and novel antibiotics discovery.
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http://dx.doi.org/10.1038/s41598-021-86997-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041823PMC
April 2021

Deciphering the Binding Interactions between ACP and β-ketoacyl ACP Synthase III to Improve Antibiotic Targeting Using NMR Spectroscopy.

Int J Mol Sci 2021 Mar 24;22(7). Epub 2021 Mar 24.

Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea.

Fatty acid synthesis is essential for bacterial viability. Thus, fatty acid synthases (FASs) represent effective targets for antibiotics. Nevertheless, multidrug-resistant bacteria, including the human opportunistic bacteria, are emerging threats. Meanwhile, the FAS pathway of is relatively unexplored. Considering that acyl carrier protein (ACP) has an important role in the delivery of fatty acyl intermediates to other FAS enzymes, we elucidated the solution structure of ACP (AbACP) and, using NMR spectroscopy, investigated its interactions with β-ketoacyl ACP synthase III (AbKAS III), which initiates fatty acid elongation. The results show that AbACP comprises four helices, while Ca reduces the electrostatic repulsion between acid residues, and the unconserved F47 plays a key role in thermal stability. Moreover, AbACP exhibits flexibility near the hydrophobic cavity entrance from D59 to T65, as well as in the αα loop region. Further, F29 and A69 participate in slow exchanges, which may be related to shuttling of the growing acyl chain. Additionally, electrostatic interactions occur between the α and α-helix of ACP and AbKAS III, while the hydrophobic interactions through the ACP α-helix are seemingly important. Our study provides insights for development of potent antibiotics capable of inhibiting FAS protein-protein interactions.
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http://dx.doi.org/10.3390/ijms22073317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036411PMC
March 2021

Identification of inhibitor binding hotspots in Acinetobacter baumannii β-ketoacyl acyl carrier protein synthase III using molecular dynamics simulation.

J Mol Graph Model 2020 11 8;100:107669. Epub 2020 Jul 8.

Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, South Korea. Electronic address:

Acinetobacter baumannii is a gram-negative bacterium that is rapidly developing drug resistance due to the abuse of antibiotics. The emergence of multidrug-resistant A. baumannii has greatly contributed to the urgency of developing new antibiotics. Previously, we had discovered two potent inhibitors of A. baumannii β-ketoacyl acyl carrier protein synthase III (abKAS III), YKab-4 and YKab-6, which showed potent activity against A. baumannii. In addition, we have reported the crystal structure of abKAS III. In the present study, we investigated the binding between abKAS III and its inhibitors by docking simulation. Molecular dynamics (MD) simulations were performed using docked inhibitor models to identify the hotspot residues related to inhibitor binding. The binding free energies estimated using the MD simulations suggest that residues I198 and F260 of abKAS III serve as the inhibitor binding hotspots. I198, found to be responsible for mediating hydrophobic interactions with inhibitors, had the strongest residual binding energy among all abKAS III residues. We modeled glutamine substitutions of residues I198 and F260 and estimated the relative binding energies of the I198Q and F260Q variants. The results confirmed that I198 and F260 are the key inhibitor binding residues. The roles of the key residues in inhibitor binding, i.e. F260 in the α9 helix and the I198 in the β6β7 loop region, were investigated using principal component analysis (PCA). PCA revealed the structural changes resulting from the abKAS III I198Q and F260Q mutations and described the essential dynamics of the α9 helix. In addition, the results suggest that the β6β7 loop region may act as a gate keeper for ligand binding. Hydrophobic interactions involving I198 and F260 in abKAS III appear to be essential for the binding of the inhibitors YKab-4 and YKab-6. In conclusion, this study provides valuable information for the rational design of antibiotics via the inhibition of abKAS III.
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http://dx.doi.org/10.1016/j.jmgm.2020.107669DOI Listing
November 2020

Structural Characterization of an ACP from : Insights into Hyperthermal Adaptation.

Int J Mol Sci 2020 Apr 9;21(7). Epub 2020 Apr 9.

Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea.

, a deep-branching hyperthermophilic bacterium, expresses an extraordinarily stable acyl carrier protein (-ACP) that functions as a carrier in the fatty acid synthesis system at near-boiling aqueous environments. Here, to understand the hyperthermal adaptation of -ACP, we investigated the structure and dynamics of -ACP by nuclear magnetic resonance (NMR) spectroscopy. The melting temperature of -ACP (101.4 °C) far exceeds that of other ACPs, owing to extensive ionic interactions and tight hydrophobic packing. The D59 residue, which replaces Pro/Ser of other ACPs, mediates ionic clustering between helices III and IV. This creates a wide pocket entrance to facilitate the accommodation of long acyl chains required for hyperthermal adaptation of the cell membrane. -ACP is revealed to be the first ACP that harbor an amide proton hyperprotected against hydrogen/deuterium exchange for I15. The hydrophobic interactions mediated by I15 appear to be the key driving forces of the global folding process of -ACP. Our findings provide insights into the structural basis of the hyperthermal adaptation of ACP, which might have allowed to survive in hot ancient oceans.
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http://dx.doi.org/10.3390/ijms21072600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178038PMC
April 2020

Optimal Target Assignment with Seamless Handovers for Networked Radars.

Sensors (Basel) 2019 Oct 19;19(20). Epub 2019 Oct 19.

Department of Aerospace Engineering & KI for Robotics, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea.

This paper proposes a binary linear programming formulation for multiple target assignment of a radar network and demonstrates its applicability to obtain optimal solutions using an off-the-shelf mixed-integer linear programming solver. The goal of radar resource scheduling in this paper is to assign the maximum number of targets by handing over targets between networked radar systems to overcome physical limitations such as the detection range and simultaneous tracking capability of each radar. To achieve this, time windows are generated considering the relation between each radar and target considering incoming target information. Numerical experiments using a local-scale simulation were performed to verify the functionality of the formulation and a sensitivity analysis was conducted to identify the trend of the results with respect to several parameters. Additional experiments performed for a large-scale (battlefield) scenario confirmed that the proposed formulation is valid and applicable for hundreds of targets and corresponding radar network systems composed of five distributed radars. The performance of the scheduling solutions using the proposed formulation was better than that of the general greedy algorithm as a heuristic approach in terms of objective value as well as the number of handovers.
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http://dx.doi.org/10.3390/s19204555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832481PMC
October 2019

Structural basis of branched-chain fatty acid synthesis by Propionibacterium acnes β-ketoacyl acyl Carrier protein synthase.

Biochem Biophys Res Commun 2019 01 23;509(1):322-328. Epub 2018 Dec 23.

Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea. Electronic address:

Propionibacterium acnes is an anaerobic gram-positive bacterium found in the niche of the sebaceous glands in the human skin, and is a causal pathogen of inflammatory skin diseases as well as periprosthetic joint infection. To gain effective control of P. acnes, a deeper understanding of the cellular metabolism mechanism involved in its ability to reside in this unique environment is needed. P. acnes exhibits typical cell membrane features of gram-positive bacteria, such as control of membrane fluidity by branched-chain fatty acids (BCFAs). Branching at the iso- or anteiso-position is achieved by incorporation of isobutyryl- or 2-methyl-butyryl-CoA via β-ketoacyl acyl carrier protein synthase (KAS III) from fatty acid synthesis. Here, we determined the crystal structure of P. acnes KAS III (PaKAS III) at the resolution of 1.9 Å for the first time. Conformation-sensitive urea polyacrylamide gel electrophoresis and tryptophan fluorescence quenching experiments confirmed that PaKAS III prefers isobutyryl-CoA as the acetyl-CoA, and the unique shape of the active site cavity complies with incorporation of branched-short chain CoAs. The determined structure clearly illustrates how BCFA synthesis is achieved in P. acnes. Moreover, the unique shape of the cavity required for the branched-chain primer can be invaluable in designing novel inhibitors of PaKAS III and developing new specifically targeted antibiotics.
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http://dx.doi.org/10.1016/j.bbrc.2018.12.134DOI Listing
January 2019

Elucidation of the crystal structure of FabD from the multidrug-resistant bacterium Acinetobacter baumannii.

Biochem Biophys Res Commun 2018 10 20;505(1):208-214. Epub 2018 Sep 20.

Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea. Electronic address:

Bacterial fatty acid synthesis (FAS) has been extensively studied as a potential target of antimicrobials. In FAS, FabD mediates transacylation of the malonyl group from malonyl-CoA to acyl-carrier protein (ACP). The mounting threat of nosocomial infection by multidrug-resistant Acinetobacter baumannii warrants a deeper understanding of its essential cellular mechanisms, which could lead to effective control of this highly competent pathogen. The molecular mechanisms involved in A. baumannii FAS are poorly understood, and recent research has suggested that Pseudomonas aeruginosa, a closely related nosocomial pathogen of A. baumannii, utilizes FAS to produce virulence factors. In this study, we solved the crystal structure of A. baumannii FabD (AbFabD) to provide a platform for the development of new antibacterial agents. Analysis of the structure of AbFabD confirmed the presence of highly conserved active site residues among bacterial homologs. Binding constants between AbFabD variants and A. baumannii ACP (AbACP) revealed critical conserved residues Lys195 and Lys200 involved in AbACP binding. Computational docking of a potential inhibitor, trifluoperazine, revealed a unique inhibitor-binding pocket near the substrate-binding site. The structural study presented herein will be useful for the structure-based design of potent AbFabD inhibitors.
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http://dx.doi.org/10.1016/j.bbrc.2018.09.079DOI Listing
October 2018

Microwave-assisted sintering synthesis of greenish-yellow emitting Sr SiO :Eu phosphors.

Luminescence 2018 Sep 21;33(6):1081-1086. Epub 2018 Jun 21.

Department of Physics, Changwon National University, Changwon, Republic of Korea.

Europium ion (Eu ) doped Sr SiO phosphors with greenish-yellow emission were synthesized using microwave-assisted sintering. The phase structure and photoluminescence (PL) properties of the obtained phosphor samples were investigated. The PL excitation spectra of the Sr SiO :Eu phosphors exhibited a broad band in the range of 260 nm to 485 nm with a maximum at 361 nm attributed to the 5f-4d allowed transition of the Eu ions. Under an excitation at 361 nm, the Sr SiO :Eu phosphor exhibited a greenish-yellow emission peak at 541 nm with an International-Commission-on-Illumination (CIE) chromaticity of (0.3064, 0.4772). The results suggest that the microwave-assisted sintering method is promising for the synthesis of phosphors owing to the decreased sintering time without the use of additional reductive agents.
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http://dx.doi.org/10.1002/bio.3511DOI Listing
September 2018

Structure and substrate specificity of β-ketoacyl-acyl carrier protein synthase III from Acinetobacter baumannii.

Mol Microbiol 2018 06 30;108(5):567-577. Epub 2018 Mar 30.

Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea.

Originally annotated as the initiator of fatty acid synthesis (FAS), β-ketoacyl-acyl carrier protein synthase III (KAS III) is a unique component of the bacterial FAS system. Novel variants of KAS III have been identified that promote the de novo use of additional extracellular fatty acids by FAS. These KAS III variants prefer longer acyl-groups, notably octanoyl-CoA. Acinetobacter baumannii, a clinically important nosocomial pathogen, contains such a multifunctional KAS III (AbKAS III). To characterize the structural basis of its substrate specificity, we determined the crystal structures of AbKAS III in the presence of different substrates. The acyl-group binding cavity of AbKAS III and co-crystal structure of AbKAS III and octanoyl-CoA confirmed that the cavity can accommodate acyl groups with longer alkyl chains. Interestingly, Cys264 formed a disulfide bond with residual CoA used in the crystallization, which distorted helices at the putative interface with acyl-carrier proteins. The crystal structure of KAS III in the alternate conformation can also be utilized for designing novel antibiotics.
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http://dx.doi.org/10.1111/mmi.13950DOI Listing
June 2018

Evaluation of the safety and efficiency of novel metallic implant scaler tips manufactured by the powder injection molding technique.

BMC Oral Health 2017 Jul 11;17(1):110. Epub 2017 Jul 11.

Department of Conservative Dentistry, Dental Research Institute and School of Dentistry, Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul, 03080, South Korea.

Background: Although many studies have compared the properties of ultrasonic scaling instruments, it remains controversial as to which is most suitable for implant scaling. This study evaluated the safety and efficiency of novel metallic ultrasonic scaler tips made by the powder injection molding (PIM) technique on titanium surfaces.

Methods: Mechanical instrumentation was carried out using four types of metal scaler tips consisting of copper (CU), bronze (BR), 316 L stainless steel (316 L), and conventional stainless steel (SS) tips. The instrumented surface alteration image of samples was viewed with scanning electron microscope (SEM) and surface profile of the each sample was investigated with confocal laser scanning microscopy (CLSM). Arithmetic mean roughness (Ra) and maximum height roughness (Rmax) of titanium samples were measured and dissipated power of the scaler tip was estimated for scaling efficiency.

Results: The average Ra values caused by the 316 L and SS tip were about two times higher than those of the CU and BR tips (p < 0.05). The Rmax value showed similar results. The efficiency of the SS tip was about 3 times higher than that of CU tip, the 316 L tip is about 2.7 times higher than that of CU tip, and the BR tip is about 1.2 times higher than that of CU tip.

Conclusions: Novel metallic bronze alloy ultrasonic scaler tip minimally damages titanium surfaces, similar to copper alloy tip. Therefore, this bronze alloy scaler tip may be promising instrument for implant maintenance therapy.
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http://dx.doi.org/10.1186/s12903-017-0396-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5504980PMC
July 2017

Crystal structure of dibenzothiophene sulfone monooxygenase BdsA from Bacillus subtilis WU-S2B.

Proteins 2017 06 7;85(6):1171-1177. Epub 2017 Mar 7.

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan.

The dibenzothiophene (DBT) sulfone monooxygenase BdsA from Bacillus subtilis WU-S2B catalyzes the conversion of DBT sulfone to 2'-hydroxybiphenyl 2-sulfinate. We report the crystal structures of BdsA at a resolution of 2.80 Å. BdsA exists as a homotetramer with a dimer-of-dimers configuration in the crystal, and the interaction between E288 and R296 in BdsA is important for tetramer formation. A structural comparison with homologous proteins shows that the orientation and location of the α9-α12 helices in BdsA are closer to those of the closed form than those of the open form in the EDTA monooxygenase EmoA. Proteins 2017; 85:1171-1177. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/prot.25267DOI Listing
June 2017

Insight into the transition between the open and closed conformations of Thermus thermophilus carboxypeptidase.

Biochem Biophys Res Commun 2017 03 2;484(4):787-793. Epub 2017 Feb 2.

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address:

Carboxypeptidase cleaves the C-terminal amino acid residue from proteins and peptides. Here, we report the functional and structural characterizations of carboxypeptidase belonging to the M32 family from the thermophilic bacterium Thermus thermophilus HB8 (TthCP). TthCP exhibits a relatively broad specificity for both hydrophilic (neutral and basic) and hydrophobic (aliphatic and aromatic) residues at the C-terminus and shows optimal activity in the temperature range of 75-80 °C and in the pH range of 6.8-7.2. Enzyme activity was significantly enhanced by cobalt or cadmium and was moderately inhibited by Tris at 25 °C. We also determined the crystal structure of TthCP at 2.6 Å resolution. Two dimer types of TthCP are present in the crystal. One type consists of two subunits in different states, open and closed, with a C RMSD value of 2.2 Å; the other type consists of two subunits in the same open state. This structure enables us to compare the open and closed states of an M32 carboxypeptidase. The TthCP subunit can be divided into two domains, L and S, which are separated by a substrate-binding groove. The L and S domains in the open state are almost identical to those in the closed state, with C RMSD values of 0.84 and 0.53 Å, respectively, suggesting that the transition between the open and closed states proceeds with a large hinge-bending motion. The superimposition between the closed states of TthCP and BsuCP, another M32 family member, revealed that most putative substrate-binding residues in the grooves are oriented in the same direction.
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http://dx.doi.org/10.1016/j.bbrc.2017.01.167DOI Listing
March 2017

Collaborative Management of Combined Periodontal-endodontic Lesions with a Palatogingival Groove: A Case Series.

J Endod 2017 Feb 15;43(2):332-337. Epub 2016 Dec 15.

Department of Periodontology, School of Dentistry and Dental Research Institute, BK21 Program, Seoul National University, Seoul, Korea. Electronic address:

Introduction: This article reports 3 representative cases of interdisciplinary management of a palatogingival groove in maxillary lateral incisors. The development, pathology, and effectiveness of management approaches in cases involving a combined periodontal-endodontic lesion with a palatogingival groove are discussed.

Methods: We describe 3 patients with a noncontributory medical history presenting with a chief complaint related to a maxillary incisor and diagnosed with a combined periodontal-endodontic lesion with a palatogingival groove at Seoul National University Dental Hospital, Seoul, Korea.

Results: Palatogingival grooves were mostly associated with deep periodontal pockets connected to a periapical lesion. Optional collaborative treatments were performed according to the condition as follows: case 1, root canal treatment (RCT), open flap debridement, odontoplasty, and guided tissue regeneration; case 2, RCT, apicoectomy, open flap debridement, and odontoplasty; and case 3, RCT, crown restoration, root planning, and odontoplasty. After clinical examination and radiographic assessments, the periapical lesion and periodontal deep pocket were successfully resolved with periodontal-endodontic collaborative treatment involving both periodontal surgical procedures (cases 1 and 2) and a nonsurgical procedure (case 3).

Conclusions: Within the limitations of this study, these case reports show that accurate diagnosis of developmental anomalies and elimination of inflammatory irritants are key factors for favorable long-term outcomes.
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http://dx.doi.org/10.1016/j.joen.2016.10.003DOI Listing
February 2017

Structure-activity relationship-based screening of antibiotics against Gram-negative Acinetobacter baumannii.

Bioorg Med Chem 2017 01 2;25(1):372-380. Epub 2016 Nov 2.

Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea. Electronic address:

To discover potent antibiotics against the Gram-negative bacteria, we performed a structure-activity relationship (SAR) study of YKsa-6, which was the most potent inhibitor of Staphylococcus aureus β-ketoacyl acyl carrier protein III in our previous study. We identified and selected 11 candidates, and finally screened two active compounds, YKab-4 (4-[(3-chloro-4-methylphenyl)aminoiminomethyl]benzene-1,3-diol) and YKab-6 (4-[[3-(trifluoromethyl)phenyl]aminoiminomethyl]phenol) as inhibitors of Acinetobacter baumannii KAS III (abKAS III). They showed potent antimicrobial activities at 2 or 8 μg/mL, specifically against Acinetobacter baumannii and a strong binding affinity for abKAS III. From the homology modeling, we defined the three-dimensional (3D) structure of abKAS III for the first time and found that it had an extra loop region compared with common Gram-negative bacteria derived KAS IIIs. The docking study revealed that the hydroxyl groups of inhibitors formed extensive hydrogen bonds and the complicated hydrophobic and cation-stacking interactions are important to binding with abKAS III. We confirmed that the hydrophobicity of these compounds might be the essential factor for their antimicrobial activities against Gram-negative bacteria as well as their structural rigidity, a cooperative feature for retaining the hydrophobic interactions between abKAS III and its inhibitors. This study may provide an insight developing strategies for potent antibiotics against A. baumannii.
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http://dx.doi.org/10.1016/j.bmc.2016.11.001DOI Listing
January 2017

The structure of the pleiotropic transcription regulator CodY provides insight into its GTP-sensing mechanism.

Nucleic Acids Res 2016 Nov 4;44(19):9483-9493. Epub 2016 Sep 4.

Department of Biosystems & Biotechnology, Korea University, Anam-dong, Seoungbuk-gu, Seoul 136-713, South Korea

GTP and branched-chain amino acids (BCAAs) are metabolic sensors that are indispensable for the determination of the metabolic status of cells. However, their molecular sensing mechanism remains unclear. CodY is a unique global transcription regulator that recognizes GTP and BCAAs as specific signals and affects expression of more than 100 genes associated with metabolism. Herein, we report the first crystal structures of the full-length CodY complex with sensing molecules and describe their functional states. We observed two different oligomeric states of CodY: a dimeric complex of CodY from Staphylococcus aureus with the two metabolites GTP and isoleucine, and a tetrameric form (apo) of CodY from Bacillus cereus Notably, the tetrameric state shows in an auto-inhibitory manner by blocking the GTP-binding site, whereas the binding sites of GTP and isoleucine are clearly visible in the dimeric state. The GTP is located at a hinge site between the long helical region and the metabolite-binding site. Together, data from structural and electrophoretic mobility shift assay analyses improve understanding of how CodY senses GTP and operates as a DNA-binding protein and a pleiotropic transcription regulator.
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http://dx.doi.org/10.1093/nar/gkw775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100569PMC
November 2016

The 1:2 complex between RavZ and LC3 reveals a mechanism for deconjugation of LC3 on the phagophore membrane.

Autophagy 2017 Jan 28;13(1):70-81. Epub 2016 Oct 28.

a Department of Life Sciences , Korea University , Seongbuk-gu, Seoul , Korea.

Hosts utilize macroautophagy/autophagy to clear invading bacteria; however, bacteria have also developed a specific mechanism to survive by manipulating the host cell autophagy mechanism. One pathogen, Legionella pneumophila, can hinder host cell autophagy by using the specific effector protein RavZ that cleaves phosphatidylethanolamine-conjugated LC3 on the phagophore membrane. However, the detailed molecular mechanisms associated with the function of RavZ have hitherto remained unclear. Here, we report on the biochemical characteristics of the RavZ-LC3 interaction, the solution structure of the 1:2 complex between RavZ and LC3, and crystal structures of RavZ showing different conformations of the active site loop without LC3. Based on our biochemical, structural, and cell-based analyses of RavZ and LC3, both distant flexible N- and C-terminal regions containing LC3-interacting region (LIR) motifs are important for substrate recognition. These results suggest a novel mechanism of RavZ action on the phagophore membrane and lay the groundwork for understanding how bacterial pathogens can survive autophagy.
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http://dx.doi.org/10.1080/15548627.2016.1243199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240826PMC
January 2017

Processing of A-form ssDNA by cryptic RNase H fold exonuclease PF2046.

Arch Biochem Biophys 2016 09 3;606:143-50. Epub 2016 Aug 3.

Division of Biotechnology, Korea University, Anam-Dong, Seongbuk-gu, Seoul 136-713, Republic of Korea; Institute for Life Sciences and Natural Resources, Korea University, Seoul 136-713, Republic of Korea. Electronic address:

RNase H fold protein PF2046 of Pyrococcus furiosus is a 3'-5' ssDNA exonuclease that cleaves after the second nucleotide from the 3' end of ssDNA and prefers poly-dT over poly-dA as a substrate. In our crystal structure of PF2046 complexed with an oligonucleotide of four thymidine nucleotides (dT4), PF2046 accommodates dT4 tightly in a groove and imposes steric hindrance on dT4 mainly by Phe220 such that dT4 assumes the A-form. As poly-dA prefer B-form due to the stereochemical restrictions, the A-form ssDNA binding by PF2046 should disfavor the processing of poly-dA. Phe220 variants display reduced activity toward poly-dA and the A-form appears to be a prerequisite for the processing by PF2046.
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http://dx.doi.org/10.1016/j.abb.2016.08.001DOI Listing
September 2016

Structural identification of the lipopolysaccharide-binding capability of a cupin-family protein from Helicobacter pylori.

FEBS Lett 2016 09 11;590(17):2997-3004. Epub 2016 Aug 11.

Department of Biotechnology, Research Institute (RIBHS) and College of Biomedical and Health Science, Konkuk University, Chungbuk, Korea.

We solved the crystal structure of a functionally uncharacterized protein, HP0902, from Helicobacter pylori. Its structure demonstrated an all-β cupin fold that cannot bind metal ions due to the absence of a metal-binding histidine that is conserved in many metallo-cupins. In contrast, isothermal titration calorimetry and NMR titration demonstrated that HP0902 is able to bind bacterial endotoxin lipopolysaccharides (LPS) through its surface-exposed loops, where metal-binding sites are usually found in other metallo-cupins. This report constitutes the first identification of an LPS-interacting protein, both in the cupin family and in H. pylori. Furthermore, identification of the ability of HP0902 to bind LPS uncovers a putative role for this protein in H. pylori pathogenicity.
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http://dx.doi.org/10.1002/1873-3468.12332DOI Listing
September 2016

Evaluation of dentin tubule occlusion after laser irradiation and desensitizing agent application.

Am J Dent 2015 Oct;28(5):303-8

Purpose: To evaluate the effects of lasers (Nd:YAG and Er:YAG) and of topical desensitizing agents on dentin tubule occlusion by measuring real-time dentin fluid flow (DFF).

Methods: 32 molars were prepared with V-shape cavity at the cervical area, acid-etched, water rinsed, blotted dry, and treated with (1) Nd:YAG laser; (2) Er:YAG laser; (3) SuperSeal, a desensitizing agent; (4) ClinproXT, a resin-modified glass-ionomer (RMGI) varnish (n = 8 each). A real-time fluid flow measuring instrument (nano-Flow) was used to measure the DFF throughout the procedures. The DFF rates before and after the treatment were compared. Moreover, the surface topography of dentin tubules after each desensitizing method was examined using SEM.

Results: DFF varied among the groups. The DFF rate was significantly reduced after laser irradiation/application of the desensitizing agents (P < 0.05). ClinproXT showed the greatest reduction of DFF rate (71.9%), followed by the SuperSeal (34.8%) and laser groups (P< 0.05). However, there was no significant difference between the Nd:YAG (24.1%) and Er:YAG (20.6%) groups (P > 0.05). In SEM images, narrowed dentin tubules were observed in both lased groups and SuperSeal group. In the ClinproXT group, the occluded dentin tubules by the RMGI covering were observed.
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October 2015

The Anti-Inflammatory Effect of Human Telomerase-Derived Peptide on P. gingivalis Lipopolysaccharide-Induced Inflammatory Cytokine Production and Its Mechanism in Human Dental Pulp Cells.

Mediators Inflamm 2015 28;2015:385127. Epub 2015 Oct 28.

Department of Conservative Dentistry, Dental Research Institute and School of Dentistry, Seoul National University, Jongno-gu, Yeongun-dong 275-1, Seoul 110-749, Republic of Korea.

Porphyromonas gingivalis is considered with inducing pulpal inflammation and has lipopolysaccharide (LPS) as an inflammatory stimulator. GV1001 peptide has anticancer and anti-inflammation activity due to inhibiting activation of signaling molecules after penetration into the various types of cells. Therefore, this study examined inhibitory effect of GV1001 on dental pulp cells (hDPCs) stimulated by P. gingivalis LPS. The intracellular distribution of GV1001 was analyzed by confocal microscopy. Real-time RT-PCR was performed to determine the expression levels of TNF-α and IL-6 cytokines. The role of signaling by MAP kinases (ERK and p38) was explored using Western blot analysis. The effect of GV1001 peptide on hDPCs viability was measured by MTT assay. GV1001 was predominantly located in hDPC cytoplasm. The peptide inhibited P. gingivalis LPS-induced TNF-α and IL-6 production in hDPCs without significant cytotoxicity. Furthermore, GV1001 treatment markedly inhibited the phosphorylation of MAP kinases (ERK and p38) in LPS-stimulated hDPCs. GV1001 may prevent P. gingivalis LPS-induced inflammation of apical tissue. Also, these findings provide mechanistic insight into how GV1001 peptide causes anti-inflammatory actions in LPS-stimulated pulpitis without significantly affecting cell viability.
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http://dx.doi.org/10.1155/2015/385127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641190PMC
September 2016

Crystal structure of Legionella pneumophila type IV secretion system effector LegAS4.

Biochem Biophys Res Commun 2015 Oct 24;465(4):817-24. Epub 2015 Aug 24.

Division of Biotechnology, Korea University, Anam-Dong, Seongbuk-Gu, Seoul 136-713, Republic of Korea; Institute of Life Science and Natural Resources, Korea University, Seoul 136-713, Republic of Korea. Electronic address:

The SET domain of LegAS4, a type IV secretion system effector of Legionella pneumophila, is a eukaryotic protein motif involved in histone methylation and epigenetic modulation. The SET domain of LegAS4 is involved in the modification of Lys4 of histone H3 (H3K4) in the nucleolus of the host cell, thereby enhancing heterochromatic rDNA transcription. Moreover, LegAS4 contains an ankyrin repeat domain of unknown function at its C-terminal region. Here, we report the crystal structure of LegAS4 in complex with S-adenosyl-l-methionine (SAM). Our data indicate that the ankyrin repeats interact extensively with the SET domain, especially with the SAM-binding amino acids, through conserved residues. Conserved surface analysis marks Glu159, Glu203, and Glu206 on the SET domain serve as candidate residues involved in interaction with the positively charged histone tail. Conserved surface residues on the ankyrin repeat domain surround a small pocket, which is suspected to serve as a binding site for an unknown ligand.
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http://dx.doi.org/10.1016/j.bbrc.2015.08.094DOI Listing
October 2015

Crystal structures of apo-DszC and FMN-bound DszC from Rhodococcus erythropolis D-1.

FEBS J 2015 Aug 11;282(16):3126-35. Epub 2015 Feb 11.

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan.

Unlabelled: The release of SO2 from petroleum products derived from crude oil, which contains sulfur compounds such as dibenzothiophene (DBT), leads to air pollution. The '4S' metabolic pathway catalyzes the sequential conversion of DBT to 2-hydroxybiphenyl via three enzymes encoded by the dsz operon in several bacterial species. DszC (DBT monooxygenase), from Rhodococcus erythropolis D-1 is involved in the first two steps of the '4S' pathway. Here, we determined the first crystal structure of FMN-bound DszC, and found that two distinct conformations occur in the loop region (residues 131-142) adjacent to the active site. On the basis of the DszC-FMN structure and the previously reported apo structures of DszC homologs, the binding site for DBT and DBT sulfoxide is proposed.

Database: The atomic coordinates and structure factors for apo-DszC (PDB code: 3X0X) and DszC-FMN (PDB code: 3X0Y) have been deposited in the Protein Data Bank (http://www.rcsb.org).
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http://dx.doi.org/10.1111/febs.13216DOI Listing
August 2015

Effect from surface treatment of nickel-titanium rotary files on the fracture resistance.

Scanning 2015 Jan-Feb;37(1):82-7. Epub 2014 Dec 8.

Department of Conservative Dentistry, School of Dentistry, Dental Research Institute, Pusan National University, Yangsan, Korea.

This study was aimed to compare the cyclic fatigue resistance and torsional resistance of rotary instruments with and without surface treatment. G6 A2 (Group A2) with and G6 A2 without surface treatment after machining (Group AN) were compared in this study. ProTaper F2 (Group F2) which has similar dimension and shape was also used for comparison. To evaluate the torsional resistance, ultimate torsional strength and distortion angle until fracture were recorded, and the toughness was calculated. The cyclic fatigue resistance was compared by evaluating the number of cycles to failure in a simulated canal. Statistical analysis was performed by one-way analysis of variance and Tukey post hoc test (p = 0.05). After torsional and cyclic fatigue tests, all fracture fragments were observed under a scanning electron microscope. Group A2 showed higher cyclic fatigue resistance than the groups AN and F2 (p < 0.05). Although group A2 demonstrated lower ultimate torsional strength than the others, there were no significant differences in toughness among the groups. While obvious machining grooves were seen in groups AN and F2, group A2 showed smooth surface resulting from the surface treatment. The specimens of fracture fragments showed typical features of cyclic failure such as micro-cracks, overloaded fast fracture zone, and torsional fracture such as unwinding helix, circular abrasion marks and dimples. Under the conditions of this study, the surface treated instruments may improve cyclic fatigue resistance while maintaining the torsional resistances and mechanical properties.
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http://dx.doi.org/10.1002/sca.21182DOI Listing
May 2015

A new class of peptidomimetics targeting the polo-box domain of Polo-like kinase 1.

J Med Chem 2015 Jan 5;58(1):294-304. Epub 2014 Nov 5.

Division of Magnetic Resonance, Korea Basic Science Institute , 804-1, Yangcheong Ri, Ochang, Chungbuk, Cheongwon 363-883, Republic of Korea.

Recent progress in the development of peptide-derived Polo-like kinase (Plk1) polo-box domain (PBD) inhibitors has led to the synthesis of multiple peptide ligands with high binding affinity and selectivity. However, few systematic analyses have been conducted to identify key Plk1 residues and characterize their interactions with potent Plk1 peptide inhibitors. We performed systematic deletion analysis using the most potent 4j peptide and studied N-terminal capping of the minimal peptide with diverse organic moieties, leading to the identification of the peptidomimetic 8 (AB-103) series with high binding affinity and selectivity. To evaluate the bioavailability of short peptidomimetic ligands, PEGylated 8 series were synthesized and incubated with HeLa cells to test for cellular uptake, antiproliferative activity, and Plk1 kinase inhibition. Finally, crystallographic studies of the Plk1 PBD in complex with peptidomimetics 8 and 22 (AB-103-5) revealed the presence of two hydrogen bond interactions responsible for their high binding affinity and selectivity.
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http://dx.doi.org/10.1021/jm501147gDOI Listing
January 2015

Mechanical properties and microstructure analysis of mineral trioxide aggregate mixed with hydrophilic synthetic polymer.

J Biomed Mater Res B Appl Biomater 2015 May 8;103(4):777-82. Epub 2014 Aug 8.

Department of Conservative Dentistry, Dental Research Institute and School of Dentistry, Seoul National University, Seoul, Korea.

Introduction: In dentistry, mineral trioxide aggregate (MTA) has been widely used for root perforation, retrograde filling, pulp capping and regenerative endodontics. Despite its superior sealing ability and biocompatibility, MTA has critical drawbacks regarding handling property such as sandy property, lacking cohesive properties and wash-out tendency. So, it is necessary to improve the fluidity of MTA in order to improve its handling properties. In this study, we applied modified liquid to improve handling properties of MTA.

Methods: Polyvinyl alcohol (PVA; 3 and 5 wt %) aqueous solutions were prepared and the samples were divided into three groups: DW group (MTA mixed with distilled water), P3 group (MTA mixed with 3% PVA), and P5 group (MTA mixed with 5% PVA). Handling property, initial setting time, and compressive strength were evaluated. The microstructures were observed by Field emission scanning electron microscope (FE-SEM) and X-ray diffractometer (XRD) phase analyses were performed.

Results: PVA modified group showed similar behavior of IRM compared to DW group. The initial setting time of P3 or P5 group was significantly longer than that of DW group (p < 0.05). The compressive strength of DW group was higher than that of P3 or P5 groups (p < 0.05). Experimental groups (P3 and P5) showed no microstructural differences compared with DW group when the fractured surfaces were observed by FE-SEM with XRD patterns after 3 and 14 days.

Conclusions: Polyvinyl alcohol, a modified liquid for MTA, improved the handling properties of the material without violating its microstructure.
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http://dx.doi.org/10.1002/jbm.b.33257DOI Listing
May 2015

Comparison of the rheological properties of four root canal sealers.

Int J Oral Sci 2015 Mar 23;7(1):56-61. Epub 2015 Mar 23.

Department of Conservative Dentistry, Dental Research Institute, School of Dentistry, Seoul National University Dental Hospital, Seoul National University, Seoul, Korea.

The flowability of a root canal sealer is clinically important because it improves the penetration of the sealer into the complex root canal system. The purpose of this study was to compare the flowabilities of four root canal sealers, measured using the simple press method (ISO 6876), and their viscosities, measured using a strain-controlled rheometer. A newly developed, calcium phosphate-based root canal sealer (Capseal) and three commercial root canal sealers (AH Plus, Sealapex and Pulp Canal Sealer EWT) were used in this study. The flowabilities of the four root canal sealers were measured using the simple press method (n=5) and their viscosities were measured using a strain-controlled rheometer (n=5). The correlation between these two values was statistically analysed using Spearman's correlation test. The flow diameters and the viscosities of the root canal sealers were strongly negatively correlated (ρ=-0.8618). The viscosity of Pulp Canal Sealer EWT was the lowest and increased in the following order: AH Plus
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http://dx.doi.org/10.1038/ijos.2014.33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817532PMC
March 2015

Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling pathway.

Acta Crystallogr D Biol Crystallogr 2014 Jul 29;70(Pt 7):1944-53. Epub 2014 Jun 29.

College of Pharmacy, Korea University, Sejong-ro, Sejong 339-700, Republic of Korea.

Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1-RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1-RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432-Lys437), which correspond to the short N-terminal 3₁₀-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1-RASSF5 complex showed a longer helical structure (Ser438-Lys480) than that in the MST1 homodimer (Val441-Lys480). Moreover, extensive polar and nonpolar contacts in the MST1-RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST-RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1-RASSF5 SARAH domain in apoptosis signalling.
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http://dx.doi.org/10.1107/S139900471400947XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089488PMC
July 2014

Exploring the binding nature of pyrrolidine pocket-dependent interactions in the polo-box domain of polo-like kinase 1.

PLoS One 2013 6;8(11):e80043. Epub 2013 Nov 6.

Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chung-Buk, Republic of Korea.

Background: Over the years, a great deal of effort has been focused on the design and synthesis of potent, linear peptide inhibitors targeting the polo-like kinase 1 (Plk1), which is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its intracellular anchoring sites via its polo-box domain, and inhibiting the Plk1 polo-box domain has been considered as an approach to circumvent the specificity problems associated with inhibiting the conserved adenosine triphosphate-binding pocket. The polo-box domain consists of two different binding regions, such as the unique, broader pyrrolidine-binding pocket and the conserved, narrow, Tyr-rich hydrophobic channel, among the three Plk polo-box domains (Plks 1-3), respectively. Therefore, the studies that provide insights into the binding nature of the unique, broader pyrrolidine-binding pocket might lead to the development of selective Plk1-inhibitory compounds.

Methodology/principal Findings: In an attempt to retain the monospecificity by targeting the unique, broader pyrrolidine-binding pocket, here, for the first time, a systematic approach was undertaken to examine the structure-activity relationship of N-terminal-truncated PLHSpTM derivatives, to apply a site-directed ligand approach using bulky aromatic and non-aromatic systems, and to characterize the binding nature of these analogues using X-ray crystallographic studies. We have identified a new mode of binding interactions, having improved binding affinity and retaining the Plk1 polo-box domain specificity, at the pyrrolidine-binding pocket. Furthermore, our data revealed that the pyrrolidine-binding pocket was very specific to recognize a short and bulky hydrophobic ligand like adamantane, whereas the Tyr-rich hydrophobic channel was specific with lengthy and small hydrophobic groups.

Conclusion/significance: The progress made using our site-directed ligands validated this approach to specifically direct the ligand into the unique pyrrolidine-binding region, and it extends the applicability of the strategy for discovering selective protein-protein interaction inhibitors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080043PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3819306PMC
June 2014

The structure of brazzein, a sweet-tasting protein from the wild African plant Pentadiplandra brazzeana.

Acta Crystallogr D Biol Crystallogr 2013 Apr 14;69(Pt 4):642-7. Epub 2013 Mar 14.

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

Brazzein is the smallest sweet-tasting protein and was isolated from the wild African plant Pentadiplandra brazzeana. The brazzein molecule consists of 54 amino-acid residues and four disulfide bonds. Here, the first crystal structure of brazzein is reported at 1.8 Å resolution and is compared with previously reported solution structures. Despite the overall structural similarity, there are several remarkable differences between the crystal and solution structures both in their backbone folds and side-chain conformations. Firstly, there is an additional α-helix in the crystal structure. Secondly, the atomic r.m.s.d.s between the corresponding C(α)-atom pairs are as large as 2.0-2.2 Å between the crystal and solution structures. Thirdly, the crystal structure exhibits a molecular shape that is similar but not identical to the solution structures. The crystal structure of brazzein reported here will provide additional information and further insights into the intermolecular interaction of brazzein with the sweet-taste receptor.
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http://dx.doi.org/10.1107/S0907444913001005DOI Listing
April 2013
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