Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

  1. Tino Pleiner
  2. Mark Bates
  3. Sergei Trakhanov
  4. Chung-Tien Lee
  5. Jan Erik Schliep
  6. Hema Chug
  7. Marc Böhning
  8. Holger Stark
  9. Henning Urlaub
  10. Dirk Görlich  Is a corresponding author
  1. Max Planck Institute for Biophysical Chemistry, Germany
  2. Max Planck Institute for Biophysical Chemistry,, Germany

Abstract

Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks - each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.

Article and author information

Author details

  1. Tino Pleiner

    Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Mark Bates

    Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Sergei Trakhanov

    Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Chung-Tien Lee

    Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Jan Erik Schliep

    3D Electron Cryo-Microscopy Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Hema Chug

    Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Marc Böhning

    Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Holger Stark

    3D Electron Cryo-Microscopy Group, Max Planck Institute for Biophysical Chemistry,, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Henning Urlaub

    Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Dirk Görlich

    Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    For correspondence
    goerlich@mpibpc.mpg.de
    Competing interests
    The authors declare that no competing interests exist.

Copyright

© 2015, Pleiner et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 17,911
    views
  • 3,645
    downloads
  • 188
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Tino Pleiner
  2. Mark Bates
  3. Sergei Trakhanov
  4. Chung-Tien Lee
  5. Jan Erik Schliep
  6. Hema Chug
  7. Marc Böhning
  8. Holger Stark
  9. Henning Urlaub
  10. Dirk Görlich
(2015)
Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation
eLife 4:e11349.
https://doi.org/10.7554/eLife.11349

Share this article

https://doi.org/10.7554/eLife.11349

Further reading

    1. Biochemistry and Chemical Biology
    Emily L Dearlove, Chatrin Chatrin ... Danny T Huang
    Research Article

    Ubiquitination typically involves covalent linking of ubiquitin (Ub) to a lysine residue on a protein substrate. Recently, new facets of this process have emerged, including Ub modification of non-proteinaceous substrates like ADP-ribose by the DELTEX E3 ligase family. Here, we show that the DELTEX family member DTX3L expands this non-proteinaceous substrate repertoire to include single-stranded DNA and RNA. Although the N-terminal region of DTX3L contains single-stranded nucleic acid binding domains and motifs, the minimal catalytically competent fragment comprises the C-terminal RING and DTC domains (RD). DTX3L-RD catalyses ubiquitination of the 3’-end of single-stranded DNA and RNA, as well as double-stranded DNA with a 3’ overhang of two or more nucleotides. This modification is reversibly cleaved by deubiquitinases. NMR and biochemical analyses reveal that the DTC domain binds single-stranded DNA and facilitates the catalysis of Ub transfer from RING-bound E2-conjugated Ub. Our study unveils the direct ubiquitination of nucleic acids by DTX3L, laying the groundwork for understanding its functional implications.