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Novel gold labeling reagent to localize protein in macromolecular assemblies
Author(s) -
Anthony Kelsey C,
You Changjiang,
Piehler Jacob,
Krummel Daniel Pomeranz
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1006.2
Subject(s) - reagent , tris , chemistry , linker , nitrilotriacetic acid , colloidal gold , macromolecule , conjugate , combinatorial chemistry , protein subunit , affinity label , protein tag , biophysics , binding site , nanoparticle , biochemistry , nanotechnology , recombinant dna , chelation , materials science , biology , organic chemistry , fusion protein , mathematical analysis , mathematics , computer science , gene , operating system
Electron microscopy (EM) is a leading technique to elucidate structure of macromolecular assemblies. Obtaining high‐resolution information by EM is often hindered by sample compositional and conformational heterogeneity. It is challenging and error prone to build an informative model into a low‐resolution 3‐dimensional reconstruction. Thus, there is demand to expand upon current approaches used for accurate protein localization to aid model building. We report use of a labeling reagent consisting of tris‐nitrilotriacetic acid (tris‐NTA) conjugated with a monofunctionalized gold nanoparticle that site‐specifically labels protein termini fused to a His‐tag. Multivalent binding of tris‐NTA to a His‐tag via complexed Ni(II) ion results into subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of a protein is further ensured by the reagents conformationally restricted linker and monofunctionalized small Au nanoparticle. We have employed the label to localize His‐tagged proteins in a T. thermophiles ATPase, RuvB, and in the E. coli 50S ribosomal subunit. Our mono‐tris‐NTA‐Au conjugate specifically binds to the target protein in these assemblies, exhibiting stoichiometric labeling. Our new Au labeling reagent improves upon current labeling approaches and should have extensive applicability in sites‐pecific labeling of His‐tag protein termini in macromolecular assemblies.