Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling

Shigekazu Tabata, Marijo Jevtic, Nobutaka Kurashige, Hirokazu Fuchida, Munetsugu Kido, Kazushi Tani, Naoki Zenmyo, Shohei Uchinomiya, Harumi Harada, Makoto Itakura, Itaru Hamachi, Ryuichi Shigemoto, Akio Ojida

研究成果: Contribution to journalArticle査読

抄録

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.

本文言語英語
ページ(範囲)256-268
ページ数13
ジャーナルiScience
22
DOI
出版ステータス出版済み - 12 20 2019

All Science Journal Classification (ASJC) codes

  • General

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