RESUMO
We report the discovery of a highly reactive peptide tag for the specific cysteine conjugation of proteins. Screening of cysteine-containing peptides using ELISA-type screening yielded a 19-amino acid tag (DCPPPDDAADDAADDAADD), named DCP3 tag, which enabled the rapid and selective labeling of the tag-fused protein with a synthetic zinc complex on the surface of living cells.
Assuntos
Cisteína/química , Imagem Óptica , Peptídeos/química , Proteínas/análise , Sequência de Aminoácidos , Complexos de Coordenação/química , Ensaio de Imunoadsorção Enzimática/métodos , Células HEK293 , Humanos , Proteínas Ligantes de Maltose/análise , Imagem Óptica/métodos , Receptores Acoplados a Proteínas G/análise , Zinco/químicaRESUMO
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.