--- _id: '6659' abstract: - lang: eng text: Chemical labeling of proteins with synthetic molecular probes offers the possibility to probe the functions of proteins of interest in living cells. However, the methods for covalently labeling targeted proteins using complementary peptide tag-probe pairs are still limited, irrespective of the versatility of such pairs in biological research. Herein, we report the new CysHis tag-Ni(II) probe pair for the specific covalent labeling of proteins. A broad-range evaluation of the reactivity profiles of the probe and the CysHis peptide tag afforded a tag-probe pair with an optimized and high labeling selectivity and reactivity. In particular, the labeling specificity of this pair was notably improved compared to the previously reported one. This pair was successfully utilized for the fluorescence imaging of membrane proteins on the surfaces of living cells, demonstrating its potential utility in biological research. acknowledgement: his work was supported by the Grant-in-Aid for Scientific Research B (JSPS KAKENHI grant no. JP17H03090 to A. O.); the Scientific Research on Innovative Areas “Chemistry for Multimolecular Crowding Biosystems” (JSPS KAKENHI grant no. JP17H06349 to A. O.); and the European Union (European Research Council Advanced grant no. 694539 and Human Brain Project Ref. 720270 to R. S.). A. O. acknowledges the financial support of the Takeda Science Foundation. article_processing_charge: No article_type: original author: - first_name: Naoki full_name: Zenmyo, Naoki last_name: Zenmyo - first_name: Hiroki full_name: Tokumaru, Hiroki last_name: Tokumaru - first_name: Shohei full_name: Uchinomiya, Shohei last_name: Uchinomiya - first_name: Hirokazu full_name: Fuchida, Hirokazu last_name: Fuchida - first_name: Shigekazu full_name: Tabata, Shigekazu id: 4427179E-F248-11E8-B48F-1D18A9856A87 last_name: Tabata - first_name: Itaru full_name: Hamachi, Itaru last_name: Hamachi - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 - first_name: Akio full_name: Ojida, Akio last_name: Ojida citation: ama: Zenmyo N, Tokumaru H, Uchinomiya S, et al. Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins. Bulletin of the Chemical Society of Japan. 2019;92(5):995-1000. doi:10.1246/bcsj.20190034 apa: Zenmyo, N., Tokumaru, H., Uchinomiya, S., Fuchida, H., Tabata, S., Hamachi, I., … Ojida, A. (2019). Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins. Bulletin of the Chemical Society of Japan. Bulletin of the Chemical Society of Japan. https://doi.org/10.1246/bcsj.20190034 chicago: Zenmyo, Naoki, Hiroki Tokumaru, Shohei Uchinomiya, Hirokazu Fuchida, Shigekazu Tabata, Itaru Hamachi, Ryuichi Shigemoto, and Akio Ojida. “Optimized Reaction Pair of the CysHis Tag and Ni(II)-NTA Probe for Highly Selective Chemical Labeling of Membrane Proteins.” Bulletin of the Chemical Society of Japan. Bulletin of the Chemical Society of Japan, 2019. https://doi.org/10.1246/bcsj.20190034. ieee: N. Zenmyo et al., “Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins,” Bulletin of the Chemical Society of Japan, vol. 92, no. 5. Bulletin of the Chemical Society of Japan, pp. 995–1000, 2019. ista: Zenmyo N, Tokumaru H, Uchinomiya S, Fuchida H, Tabata S, Hamachi I, Shigemoto R, Ojida A. 2019. Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins. Bulletin of the Chemical Society of Japan. 92(5), 995–1000. mla: Zenmyo, Naoki, et al. “Optimized Reaction Pair of the CysHis Tag and Ni(II)-NTA Probe for Highly Selective Chemical Labeling of Membrane Proteins.” Bulletin of the Chemical Society of Japan, vol. 92, no. 5, Bulletin of the Chemical Society of Japan, 2019, pp. 995–1000, doi:10.1246/bcsj.20190034. short: N. Zenmyo, H. Tokumaru, S. Uchinomiya, H. Fuchida, S. Tabata, I. Hamachi, R. Shigemoto, A. Ojida, Bulletin of the Chemical Society of Japan 92 (2019) 995–1000. date_created: 2019-07-21T21:59:16Z date_published: 2019-05-15T00:00:00Z date_updated: 2021-01-12T08:08:26Z day: '15' ddc: - '570' department: - _id: RySh doi: 10.1246/bcsj.20190034 ec_funded: 1 file: - access_level: open_access checksum: 186de511d6e0ca93f5d981e2443eb8cd content_type: application/pdf creator: dernst date_created: 2020-10-02T08:49:58Z date_updated: 2020-10-02T08:49:58Z file_id: '8594' file_name: 2019_BCSJ_Zenmyo.pdf file_size: 2464903 relation: main_file success: 1 file_date_updated: 2020-10-02T08:49:58Z has_accepted_license: '1' intvolume: ' 92' issue: '5' language: - iso: eng month: '05' oa: 1 oa_version: Published Version page: 995-1000 project: - _id: 25CA28EA-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '694539' name: 'In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour' publication: Bulletin of the Chemical Society of Japan publication_identifier: issn: - '00092673' publication_status: published publisher: Bulletin of the Chemical Society of Japan quality_controlled: '1' scopus_import: '1' status: public title: Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 92 year: '2019' ... --- _id: '7391' abstract: - lang: eng text: Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM. article_processing_charge: No article_type: original author: - first_name: Shigekazu full_name: Tabata, Shigekazu id: 4427179E-F248-11E8-B48F-1D18A9856A87 last_name: Tabata - first_name: Marijo full_name: Jevtic, Marijo id: 4BE3BC94-F248-11E8-B48F-1D18A9856A87 last_name: Jevtic - first_name: Nobutaka full_name: Kurashige, Nobutaka last_name: Kurashige - first_name: Hirokazu full_name: Fuchida, Hirokazu last_name: Fuchida - first_name: Munetsugu full_name: Kido, Munetsugu last_name: Kido - first_name: Kazushi full_name: Tani, Kazushi last_name: Tani - first_name: Naoki full_name: Zenmyo, Naoki last_name: Zenmyo - first_name: Shohei full_name: Uchinomiya, Shohei last_name: Uchinomiya - first_name: Harumi full_name: Harada, Harumi id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87 last_name: Harada orcid: 0000-0001-7429-7896 - first_name: Makoto full_name: Itakura, Makoto last_name: Itakura - first_name: Itaru full_name: Hamachi, Itaru last_name: Hamachi - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 - first_name: Akio full_name: Ojida, Akio last_name: Ojida citation: ama: Tabata S, Jevtic M, Kurashige N, et al. Electron microscopic detection of single membrane proteins by a specific chemical labeling. iScience. 2019;22(12):256-268. doi:10.1016/j.isci.2019.11.025 apa: Tabata, S., Jevtic, M., Kurashige, N., Fuchida, H., Kido, M., Tani, K., … Ojida, A. (2019). Electron microscopic detection of single membrane proteins by a specific chemical labeling. IScience. Elsevier. https://doi.org/10.1016/j.isci.2019.11.025 chicago: Tabata, Shigekazu, Marijo Jevtic, Nobutaka Kurashige, Hirokazu Fuchida, Munetsugu Kido, Kazushi Tani, Naoki Zenmyo, et al. “Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.” IScience. Elsevier, 2019. https://doi.org/10.1016/j.isci.2019.11.025. ieee: S. Tabata et al., “Electron microscopic detection of single membrane proteins by a specific chemical labeling,” iScience, vol. 22, no. 12. Elsevier, pp. 256–268, 2019. ista: Tabata S, Jevtic M, Kurashige N, Fuchida H, Kido M, Tani K, Zenmyo N, Uchinomiya S, Harada H, Itakura M, Hamachi I, Shigemoto R, Ojida A. 2019. Electron microscopic detection of single membrane proteins by a specific chemical labeling. iScience. 22(12), 256–268. mla: Tabata, Shigekazu, et al. “Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.” IScience, vol. 22, no. 12, Elsevier, 2019, pp. 256–68, doi:10.1016/j.isci.2019.11.025. short: S. Tabata, M. Jevtic, N. Kurashige, H. Fuchida, M. Kido, K. Tani, N. Zenmyo, S. Uchinomiya, H. Harada, M. Itakura, I. Hamachi, R. Shigemoto, A. Ojida, IScience 22 (2019) 256–268. date_created: 2020-01-29T15:56:56Z date_published: 2019-12-20T00:00:00Z date_updated: 2024-03-27T23:30:13Z day: '20' ddc: - '570' department: - _id: RySh doi: 10.1016/j.isci.2019.11.025 ec_funded: 1 external_id: isi: - :000504652000020 pmid: - '31786521' file: - access_level: open_access checksum: f3e90056a49f09b205b1c4f8c739ffd1 content_type: application/pdf creator: dernst date_created: 2020-02-04T10:48:36Z date_updated: 2020-07-14T12:47:57Z file_id: '7448' file_name: 2019_iScience_Tabata.pdf file_size: 7197776 relation: main_file file_date_updated: 2020-07-14T12:47:57Z has_accepted_license: '1' intvolume: ' 22' issue: '12' language: - iso: eng month: '12' oa: 1 oa_version: Published Version page: 256-268 pmid: 1 project: - _id: 25CA28EA-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '694539' name: 'In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour' - _id: 25CBA828-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '720270' name: Human Brain Project Specific Grant Agreement 1 (HBP SGA 1) publication: iScience publication_identifier: issn: - 2589-0042 publication_status: published publisher: Elsevier quality_controlled: '1' related_material: record: - id: '11393' relation: dissertation_contains status: public scopus_import: '1' status: public title: Electron microscopic detection of single membrane proteins by a specific chemical labeling tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 volume: 22 year: '2019' ...