start-ver=1.4 cd-journal=joma no-vol=174 cd-vols= no-issue=5 article-no= start-page=451 end-page=459 dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230801 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Direct binding of calmodulin to the cytosolic C-terminal regions of sweet/umami taste receptors en-subtitle= kn-subtitle= en-abstract= kn-abstract=Sweet and umami taste receptors recognize chemicals such as sugars and amino acids on their extracellular side and transmit signals into the cytosol of the taste cell. In contrast to ligands that act on the extracellular side of these receptors, little is known regarding the molecules that regulate receptor functions within the cytosol. In this study, we analysed the interaction between sweet and umami taste receptors and calmodulin, a representative Ca2+-dependent cytosolic regulatory protein. High prediction scores for calmodulin binding were observed on the C-terminal cytosolic side of mouse taste receptor type 1 subunit 3 (T1r3), a subunit that is common to both sweet and umami taste receptors. Pull-down assay and surface plasmon resonance analyses showed different affinities of calmodulin to the C-terminal tails of distinct T1r subtypes. Furthermore, we found that T1r3 and T1r2 showed the highest and considerable binding to calmodulin, whereas T1r1 showed weaker binding affinity. Finally, the binding of calmodulin to T1rs was consistently higher in the presence of Ca2+ than in its absence. The results suggested a possibility of the Ca2+-dependent feedback regulation process of sweet and umami taste receptor signaling by calmodulin. en-copyright= kn-copyright= en-aut-name=YoshidaAtsuki en-aut-sei=Yoshida en-aut-mei=Atsuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=ItoAyumi en-aut-sei=Ito en-aut-mei=Ayumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=YasuiNorihisa en-aut-sei=Yasui en-aut-mei=Norihisa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=YamashitaAtsuko en-aut-sei=Yamashita en-aut-mei=Atsuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=calmodulin kn-keyword=calmodulin en-keyword=cytosol kn-keyword=cytosol en-keyword=sweet taste kn-keyword=sweet taste en-keyword=taste receptor type 1 kn-keyword=taste receptor type 1 en-keyword=umami taste kn-keyword=umami taste END start-ver=1.4 cd-journal=joma no-vol=19 cd-vols= no-issue=3 article-no= start-page=e0300981 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2024 dt-pub=20240322 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Chemical range recognized by the ligand-binding domain in a representative amino acid-sensing taste receptor, T1r2a/T1r3, from medaka fish en-subtitle= kn-subtitle= en-abstract= kn-abstract=Taste receptor type 1 (T1r) proteins are responsible for recognizing nutrient chemicals in foods. In humans, T1r2/T1r3 and T1r1/T1r3 heterodimers serve as the sweet and umami receptors that recognize sugars or amino acids and nucleotides, respectively. T1rs are conserved among vertebrates, and T1r2a/T1r3 from medaka fish is currently the only member for which the structure of the ligand-binding domain (LBD) has been solved. T1r2a/T1r3 is an amino acid receptor that recognizes various l-amino acids in its LBD as observed with other T1rs exhibiting broad substrate specificities. Nevertheless, the range of chemicals that are recognized by T1r2a/T1r3LBD has not been extensively explored. In the present study, the binding of various chemicals to medaka T1r2a/T1r3LBD was analyzed. A binding assay for amino acid derivatives verified the specificity of this protein to l-alpha-amino acids and the importance of alpha-amino and carboxy groups for receptor recognition. The results further indicated the significance of the alpha-hydrogen for recognition as replacing it with a methyl group resulted in a substantially decreased affinity. The binding ability to the protein was not limited to proteinogenic amino acids, but also to non-proteinogenic amino acids, such as metabolic intermediates. Besides l-alpha-amino acids, no other chemicals showed significant binding to the protein. These results indicate that all of the common structural groups of alpha-amino acids and their geometry in the l-configuration are recognized by the protein, whereas a wide variety of alpha-substituents can be accommodated in the ligand binding sites of the LBDs. en-copyright= kn-copyright= en-aut-name=IshidaHikaru en-aut-sei=Ishida en-aut-mei=Hikaru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YasuiNorihisa en-aut-sei=Yasui en-aut-mei=Norihisa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=YamashitaAtsuko en-aut-sei=Yamashita en-aut-mei=Atsuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=42 cd-vols= no-issue=6 article-no= start-page=698 end-page=708 dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230922 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin en-subtitle= kn-subtitle= en-abstract= kn-abstract=Single-chain monellin (SCM) is an engineered protein that links the two chains of monellin, a naturally sweet-tasting protein. This protein is an attractive candidate for use as a sugar replacement in food and beverages and has numerous other applications. Therefore, generating SCM mutants with improved stability is an active area of research to broaden the range of its potential applications. In this study, we focused on the Cys41 residue of SCM, which is a single cysteine residue present at a structurally important position. This residue is often substituted with Ser. However, this substitution may destabilize SCM because Cys41 is buried in the hydrophobic core of the protein. Therefore, we designed mutants that substituted Ala, Val, and Leu for this residue, namely C41A, C41V, and C41L. We characterized these three mutants, SCM C41S, and wild type (WT). Differential scanning fluorimetric analysis revealed that substituting Cys41 with Ala or Val increased the thermal stability of SCM, while substitution with Ser or Leu decreased its stability. Determination of the crystal structures of SCM C41A and C41V mutants revealed that the overall structures and main chain structures around the 41st residue of both mutants were almost identical to the WT. On the other hand, the orientations of the amino acid side chains near the 41st residue differed among the SCM variants. Taken together, our results indicate that substituting Cys41 with Ala or Val increases the stability of SCM and provide insight into the structural basis of this improvement. en-copyright= kn-copyright= en-aut-name=OhnumaKyosuke en-aut-sei=Ohnuma en-aut-mei=Kyosuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YamashitaAtsuko en-aut-sei=Yamashita en-aut-mei=Atsuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=YasuiNorihisa en-aut-sei=Yasui en-aut-mei=Norihisa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= affil-num=1 en-affil=School of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=School of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=School of Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=Crystallography kn-keyword=Crystallography en-keyword=Monellin kn-keyword=Monellin en-keyword=Protein Stability kn-keyword=Protein Stability en-keyword=Recombinant Proteins kn-keyword=Recombinant Proteins END start-ver=1.4 cd-journal=joma no-vol=14 cd-vols= no-issue=1 article-no= start-page=621 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230204 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Pivotal role for S-nitrosylation of DNA methyltransferase 3B in epigenetic regulation of tumorigenesis en-subtitle= kn-subtitle= en-abstract= kn-abstract=DNA methyltransferases (DNMTs) catalyze methylation at the C5 position of cytosine with S-adenosyl-l-methionine. Methylation regulates gene expression, serving a variety of physiological and pathophysiological roles. The chemical mechanisms regulating DNMT enzymatic activity, however, are not fully elucidated. Here, we show that protein S-nitrosylation of a cysteine residue in DNMT3B attenuates DNMT3B enzymatic activity and consequent aberrant upregulation of gene expression. These genes include Cyclin D2 (Ccnd2), which is required for neoplastic cell proliferation in some tumor types. In cell-based and in vivo cancer models, only DNMT3B enzymatic activity, and not DNMT1 or DNMT3A, affects Ccnd2 expression. Using structure-based virtual screening, we discovered chemical compounds that specifically inhibit S-nitrosylation without directly affecting DNMT3B enzymatic activity. The lead compound, designated DBIC, inhibits S-nitrosylation of DNMT3B at low concentrations (IC50 <= 100nM). Treatment with DBIC prevents nitric oxide (NO)-induced conversion of human colonic adenoma to adenocarcinoma in vitro. Additionally, in vivo treatment with DBIC strongly attenuates tumor development in a mouse model of carcinogenesis triggered by inflammation-induced generation of NO. Our results demonstrate that de novo DNA methylation mediated by DNMT3B is regulated by NO, and DBIC protects against tumor formation by preventing aberrant S-nitrosylation of DNMT3B. en-copyright= kn-copyright= en-aut-name=OkudaKosaku en-aut-sei=Okuda en-aut-mei=Kosaku kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=NakaharaKengo en-aut-sei=Nakahara en-aut-mei=Kengo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=ItoAkihiro en-aut-sei=Ito en-aut-mei=Akihiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=IijimaYuta en-aut-sei=Iijima en-aut-mei=Yuta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=NomuraRyosuke en-aut-sei=Nomura en-aut-mei=Ryosuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=KumarAshutosh en-aut-sei=Kumar en-aut-mei=Ashutosh kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=FujikawaKana en-aut-sei=Fujikawa en-aut-mei=Kana kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=AdachiKazuya en-aut-sei=Adachi en-aut-mei=Kazuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=ShimadaYuki en-aut-sei=Shimada en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=FujioSatoshi en-aut-sei=Fujio en-aut-mei=Satoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=YamamotoReina en-aut-sei=Yamamoto en-aut-mei=Reina kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=TakasugiNobumasa en-aut-sei=Takasugi en-aut-mei=Nobumasa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 ORCID= en-aut-name=OnumaKunishige en-aut-sei=Onuma en-aut-mei=Kunishige kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=13 ORCID= en-aut-name=OsakiMitsuhiko en-aut-sei=Osaki en-aut-mei=Mitsuhiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=14 ORCID= en-aut-name=OkadaFutoshi en-aut-sei=Okada en-aut-mei=Futoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=15 ORCID= en-aut-name=UkegawaTaichi en-aut-sei=Ukegawa en-aut-mei=Taichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=16 ORCID= en-aut-name=TakeuchiYasuo en-aut-sei=Takeuchi en-aut-mei=Yasuo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=17 ORCID= en-aut-name=YasuiNorihisa en-aut-sei=Yasui en-aut-mei=Norihisa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=18 ORCID= en-aut-name=YamashitaAtsuko en-aut-sei=Yamashita en-aut-mei=Atsuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=19 ORCID= en-aut-name=MarusawaHiroyuki en-aut-sei=Marusawa en-aut-mei=Hiroyuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=20 ORCID= en-aut-name=MatsushitaYosuke en-aut-sei=Matsushita en-aut-mei=Yosuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=21 ORCID= en-aut-name=KatagiriToyomasa en-aut-sei=Katagiri en-aut-mei=Toyomasa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=22 ORCID= en-aut-name=ShibataTakahiro en-aut-sei=Shibata en-aut-mei=Takahiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=23 ORCID= en-aut-name=UchidaKoji en-aut-sei=Uchida en-aut-mei=Koji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=24 ORCID= en-aut-name=NiuSheng-Yong en-aut-sei=Niu en-aut-mei=Sheng-Yong kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=25 ORCID= en-aut-name=LangNhi B. en-aut-sei=Lang en-aut-mei=Nhi B. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=26 ORCID= en-aut-name=NakamuraTomohiro en-aut-sei=Nakamura en-aut-mei=Tomohiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=27 ORCID= en-aut-name=ZhangKam Y. J. en-aut-sei=Zhang en-aut-mei=Kam Y. J. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=28 ORCID= en-aut-name=LiptonStuart A. en-aut-sei=Lipton en-aut-mei=Stuart A. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=29 ORCID= en-aut-name=UeharaTakashi en-aut-sei=Uehara en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=30 ORCID= affil-num=1 en-affil=Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science kn-affil= affil-num=4 en-affil=Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=6 en-affil=Laboratory for Structural Bioinformatics, Center for Biosystems Dynamics Research, RIKEN kn-affil= affil-num=7 en-affil=Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=8 en-affil=Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=9 en-affil=Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=10 en-affil=Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=11 en-affil=Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=12 en-affil=Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=13 en-affil=Division of Experimental Pathology, Faculty of Medicine, Tottori University kn-affil= affil-num=14 en-affil=Division of Experimental Pathology, Faculty of Medicine, Tottori University kn-affil= affil-num=15 en-affil=Division of Experimental Pathology, Faculty of Medicine, Tottori University kn-affil= affil-num=16 en-affil=Department of Synthetic and Medicinal Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=17 en-affil=Department of Synthetic and Medicinal Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=18 en-affil=Laboratory of Structural Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=19 en-affil=Laboratory of Structural Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=20 en-affil=Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University kn-affil= affil-num=21 en-affil=Division of Genome Medicine, Institute of Advanced Medical Sciences, Tokushima University kn-affil= affil-num=22 en-affil=Division of Genome Medicine, Institute of Advanced Medical Sciences, Tokushima University kn-affil= affil-num=23 en-affil=Graduate School of Bioagricultural Sciences, Nagoya University kn-affil= affil-num=24 en-affil=Laboratory of Food Chemistry, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo kn-affil= affil-num=25 en-affil=Broad Institute of MIT and Harvard kn-affil= affil-num=26 en-affil=Neurodegeneration New Medicines Center, and Departments of Molecular Medicine and Neuroscience, The Scripps Research Institute kn-affil= affil-num=27 en-affil=Neurodegeneration New Medicines Center, and Departments of Molecular Medicine and Neuroscience, The Scripps Research Institute kn-affil= affil-num=28 en-affil=Laboratory for Structural Bioinformatics, Center for Biosystems Dynamics Research, RIKEN kn-affil= affil-num=29 en-affil=Neurodegeneration New Medicines Center, and Departments of Molecular Medicine and Neuroscience, The Scripps Research Institute kn-affil= affil-num=30 en-affil=Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=12 cd-vols= no-issue= article-no= start-page=e84291 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230228 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Chloride ions evoke taste sensations by binding to the extracellular ligand-binding domain of sweet/umami taste receptors en-subtitle= kn-subtitle= en-abstract= kn-abstract=Salt taste sensation is multifaceted: NaCl at low or high concentrations is preferably or aversively perceived through distinct pathways. Cl- is thought to participate in taste sensation through an unknown mechanism. Here, we describe Cl- ion binding and the response of taste receptor type 1 (T1r), a receptor family composing sweet/umami receptors. The T1r2a/T1r3 heterodimer from the medaka fish, currently the sole T1r amenable to structural analyses, exhibited a specific Cl- binding in the vicinity of the amino-acid-binding site in the ligand-binding domain (LBD) of T1r3, which is likely conserved across species, including human T1r3. The Cl- binding induced a conformational change in T1r2a/T1r3LBD at sub- to low-mM concentrations, similar to canonical taste substances. Furthermore, oral Cl- application to mice increased impulse frequencies of taste nerves connected to T1r-expressing taste cells and promoted their behavioral preferences attenuated by a T1r-specific blocker or T1r3 knock-out. These results suggest that the Cl- evokes taste sensations by binding to T1r, thereby serving as another preferred salt taste pathway at a low concentration. en-copyright= kn-copyright= en-aut-name=AtsumiNanako en-aut-sei=Atsumi en-aut-mei=Nanako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YasumatsuKeiko en-aut-sei=Yasumatsu en-aut-mei=Keiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=TakashinaYuriko en-aut-sei=Takashina en-aut-mei=Yuriko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=ItoChiaki en-aut-sei=Ito en-aut-mei=Chiaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=YasuiNorihisa en-aut-sei=Yasui en-aut-mei=Norihisa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=MargolskeeRobert F. en-aut-sei=Margolskee en-aut-mei=Robert F. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=YamashitaAtsuko en-aut-sei=Yamashita en-aut-mei=Atsuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=School of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=6 en-affil=Monell Chemical Senses Center kn-affil= affil-num=7 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=taste receptor kn-keyword=taste receptor en-keyword=salt taste kn-keyword=salt taste en-keyword=chloride kn-keyword=chloride en-keyword=O kn-keyword=O en-keyword=latipes kn-keyword=latipes en-keyword=Mouse kn-keyword=Mouse en-keyword=Other kn-keyword=Other END start-ver=1.4 cd-journal=joma no-vol=169 cd-vols= no-issue=5 article-no= start-page=585 end-page=599 dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=202112 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=A sweet protein monellin as a non-antibody scaffold for synthetic binding proteins en-subtitle= kn-subtitle= en-abstract= kn-abstract=Synthetic binding proteins that have the ability to bind with molecules can be generated using various protein domains as non-antibody scaffolds. These designer proteins have been used widely in research studies, as their properties overcome the disadvantages of using antibodies. Here, we describe the first application of a phage display to generate synthetic binding proteins using a sweet protein, monellin, as a non-antibody scaffold. Single-chain monellin (scMonellin), in which two polypeptide chains of natural monellin are connected by a short linker, has two loops on one side of the molecule. We constructed phage display libraries of scMonellin, in which the amino acid sequence of the two loops is diversified. To validate the performance of these libraries, we sorted them against the folding mutant of the green fluorescent protein variant (GFPuv) and yeast small ubiquitin-related modifier. We successfully obtained scMonellin variants exhibiting moderate but significant affinities for these target proteins. Crystal structures of one of the GFPuv-binding variants in complex with GFPuv revealed that the two diversified loops were involved in target recognition. scMonellin, therefore, represents a promising non-antibody scaffold in the design and generation of synthetic binding proteins. We termed the scMonellin-derived synthetic binding proteins ‘SWEEPins’. en-copyright= kn-copyright= en-aut-name=YasuiNorihisa en-aut-sei=Yasui en-aut-mei=Norihisa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=NakamuraKazuaki en-aut-sei=Nakamura en-aut-mei=Kazuaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=YamashitaAtsuko en-aut-sei=Yamashita en-aut-mei=Atsuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Kita-ku, Okayama 700-8530, Japan kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Kita-ku, Okayama 700-8530, Japan kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Kita-ku, Okayama 700-8530, Japan kn-affil= en-keyword=combinatorial library kn-keyword=combinatorial library en-keyword=non-antibody scaffold kn-keyword=non-antibody scaffold en-keyword=phage display kn-keyword=phage display en-keyword=single-chain monellin kn-keyword=single-chain monellin en-keyword=synthetic binding proteins kn-keyword=synthetic binding proteins END start-ver=1.4 cd-journal=joma no-vol=14 cd-vols= no-issue=10 article-no= start-page=e0218909 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2019 dt-pub=20191004 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3 en-subtitle= kn-subtitle= en-abstract= kn-abstract=Taste receptor type 1 (T1r) is responsible for the perception of essential nutrients, such as sugars and amino acids, and evoking sweet and umami (savory) taste sensations. T1r receptors recognize many of the taste substances at their extracellular ligand-binding domains (LBDs). In order to detect a wide array of taste substances in the environment, T1r receptors often possess broad ligand specificities. However, the entire ranges of chemical spaces and their binding characteristics to any T1rLBDs have not been extensively analyzed. In this study, we exploited the differential scanning fluorimetry (DSF) to medaka T1r2a/T1r3LBD, a current sole T1rLBD heterodimer amenable for recombinant preparation, and analyzed their thermal stabilization by adding various amino acids. The assay showed that the agonist amino acids induced thermal stabilization and shifted the melting temperatures (T-m) of the protein. An agreement between the DSF results and the previous biophysical assay was observed, suggesting that DSF can detect ligand binding at the orthostericbinding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested Lamino acids, but no D-amino acid, induced T-m shifts of T1r2a/T1r3LBD, indicating the broad L-amino acid specificities of the proteins probably with several different manners of recognition. The T-m shifts by each amino acid also showed a fair correlation with the responses exhibited by the full-length receptor, verifying the broad amino-acid binding profiles at the orthosteric site in LBD observed by DSF. en-copyright= kn-copyright= en-aut-name=YoshidaTakashi en-aut-sei=Yoshida en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YasuiNorihisa en-aut-sei=Yasui en-aut-mei=Norihisa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KusakabeYuko en-aut-sei=Kusakabe en-aut-mei=Yuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=ItoChiaki en-aut-sei=Ito en-aut-mei=Chiaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=AkamatsuMiki en-aut-sei=Akamatsu en-aut-mei=Miki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=YamashitaAtsuko en-aut-sei=Yamashita en-aut-mei=Atsuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Food Research Institute, National Agriculture and Food Research Organization kn-affil= affil-num=4 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Graduate School of Agriculture, Kyoto University kn-affil= affil-num=6 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=9 cd-vols= no-issue= article-no= start-page=4722 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2019 dt-pub=2019318 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Specific modification at the C-terminal lysine residue of the green fluorescent protein variant, GFPuv, expressed in Escherichia coli en-subtitle= kn-subtitle= en-abstract= kn-abstract=Green fluorescent protein (GFP) is amenable to recombinant expression in various kinds of cells and is widely used in life science research. We found that the recombinant expression of GFPuv, a commonly-used mutant of GFP, in E. coli produced two distinct molecular species as judged by in-gel fluorescence SDS-PAGE. These molecular species, namely form I and II, could be separately purified by anion-exchange chromatography without any remarkable differences in the fluorescence spectra. Mass spectrometric analyses revealed that the molecular mass of form I is almost the same as the calculated value, while that of form II is approximately 1 Da larger than that of form I. Further mass spectrometric top-down sequencing pinpointed the modification in GFPuv form II, where the epsilon-amino group of the C-terminal Lys238 residue is converted into the hydroxyl group. No equivalent modification was observed in the native GFP in jellyfish Aequorea victoria, suggesting that this modification is not physiologically relevant. Crystal structure analysis of the two species verified the structural identity of the backbone and the vicinity of the chromophore. The modification found in this study may also be generated in other GFP variants as well as in other recombinant expression systems. en-copyright= kn-copyright= en-aut-name=NakataniTakahiro en-aut-sei=Nakatani en-aut-mei=Takahiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YasuiNorihisa en-aut-sei=Yasui en-aut-mei=Norihisa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=TamuraIssei en-aut-sei=Tamura en-aut-mei=Issei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=YamashitaAtsuko en-aut-sei=Yamashita en-aut-mei=Atsuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= affil-num=1 en-affil= Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil= Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil= Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil= Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END