High-Affinity Gold Nanoparticle Pin to Label and Localize Histidine-Tagged Protein in Macromolecular Assemblies
Autor(en): | Anthony, Kelsey C. You, Changjiang Piehler, Jacob Krummel, Daniel A. Pomeranz |
Stichwörter: | BINDING; Biochemistry & Molecular Biology; Biophysics; Cell Biology; COMPLEXES; CRYSTAL-STRUCTURE; ELECTRON-MICROSCOPY; LIVE CELLS; MIGRATION MOTOR PROTEIN; PURIFICATION; RESOLUTION; RUVB; TRACKING | Erscheinungsdatum: | 2014 | Herausgeber: | CELL PRESS | Journal: | STRUCTURE | Volumen: | 22 | Ausgabe: | 4 | Startseite: | 628 | Seitenende: | 635 | Zusammenfassung: | There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle ((AuNP)tris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1: 1 stoichiometry. Precise localization of (AuNP)tris-NTA labeled proteins by electron microscopy is further ensured by the reagent's short conformationally restricted linker. We used (AuNP)tris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. (AuNP)tris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our labeling reagent should find broad application in noncovalent, site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies. |
ISSN: | 09692126 | DOI: | 10.1016/j.str.2014.01.007 |
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