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

• Published in: iScience Vol. 22; pp. 256 - 268
• Main Authors: Tabata, Shigekazu, Jevtic, Marijo, Kurashige, Nobutaka, Fuchida, Hirokazu, Kido, Munetsugu, Tani, Kazushi, Zenmyo, Naoki, Uchinomiya, Shohei, Harada, Harumi, Itakura, Makoto, Hamachi, Itaru, Shigemoto, Ryuichi, Ojida, Akio
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.12.2019; Elsevier
• Subjects: Biochemistry Methods; Nanoparticles; Structural Biology; Nanoparticles; Biochemistry Methods; Structural Biology
• ISSN: 2589-0042; 2589-0042
• DOI: 10.1016/j.isci.2019.11.025

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. [Display omitted] •Developed peptide tag/probe pair enables specific chemical labeling of proteins•Chemical labeling visualizes GPCRs on cell surface with 1.4-nm gold particles•Chemical labeling has higher spatial resolution than immunogold labeling in EM•Chemical labeling shows a few times higher efficiency than immunogold labeling Nanoparticles; Biochemistry Methods; Structural Biology