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.

Reviewing Editor

  1. Karsten Weis, ETH Zürich, Switzerland

Version history

  1. Received: September 3, 2015
  2. Accepted: December 2, 2015
  3. Accepted Manuscript published: December 3, 2015 (version 1)
  4. Accepted Manuscript updated: December 5, 2015 (version 2)
  5. Version of Record published: February 3, 2016 (version 3)
  6. Version of Record updated: March 16, 2016 (version 4)

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

  • 17,382
    views
  • 3,566
    downloads
  • 182
    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
    2. Structural Biology and Molecular Biophysics
    Marian Brenner, Christoph Zink ... Antje Gohla
    Research Article

    Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5’-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.