title: High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies.
abstract: 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.
authors.affiliation: Department of Biochemistry, Brandeis University, 415 South Street, Waltham, MA 02454, USA.
authors.forename: Kelsey C
authors.initials: KC
authors.lastname: Anthony
authors.affiliation: Department of Biology, University of Osnabrück, Barbarastraße 11, Osnabrück 49076, Germany.
authors.forename: Changjiang
authors.initials: C
authors.lastname: You
authors.affiliation: Department of Biology, University of Osnabrück, Barbarastraße 11, Osnabrück 49076, Germany. Electronic address: piehler@uos.de.
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
authors.affiliation: Department of Biochemistry, Brandeis University, 415 South Street, Waltham, MA 02454, USA. Electronic address: dapk@brandeis.edu.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Pomeranz Krummel
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D006046:Gold / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D007202:Indicators and Reagents / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry*; D008854:Microscopy, Electron; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods; D016963:Thermus thermophilus / Q000737:chemistry; D014325:Tromethamine / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins; D014325:Tromethamine; D006639:Histidine; D006046:Gold; D000251:Adenosine Triphosphatases; D009571:Nitrilotriacetic Acid