title: Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation.
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 <2 nm 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.
authors.affiliation: Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Tino
authors.identifier: http://orcid.org/0000-0002-5104-0315
authors.initials: T
authors.lastname: Pleiner
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Mark
authors.identifier: http://orcid.org/0000-0003-0668-5277
authors.initials: M
authors.lastname: Bates
authors.affiliation: Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Sergei
authors.identifier: http://orcid.org/0000-0002-1326-6153
authors.initials: S
authors.lastname: Trakhanov
authors.affiliation: Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.|Bioanalytics, Institute for Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Chung-Tien
authors.initials: CT
authors.lastname: Lee
authors.affiliation: 3D Electron Cryo-Microscopy Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Jan Erik
authors.identifier: http://orcid.org/0000-0001-7103-5954
authors.initials: JE
authors.lastname: Schliep
authors.affiliation: Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Hema
authors.identifier: http://orcid.org/0000-0002-9889-7076
authors.initials: H
authors.lastname: Chug
authors.affiliation: Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Marc
authors.identifier: http://orcid.org/0000-0002-4245-5113
authors.initials: M
authors.lastname: Böhning
authors.affiliation: 3D Electron Cryo-Microscopy Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Stark
authors.affiliation: Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.|Bioanalytics, Institute for Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Henning
authors.identifier: http://orcid.org/0000-0003-1837-5233
authors.initials: H
authors.lastname: Urlaub
authors.affiliation: Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Dirk
authors.identifier: http://orcid.org/0000-0002-4343-5210
authors.initials: D
authors.lastname: Görlich
mesh_terms: D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D046911:Macromolecular Substances / Q000276:immunology / Q000302:isolation & purification*; D028861:Nuclear Pore Complex Proteins / Q000276:immunology / Q000302:isolation & purification*; D061848:Optical Imaging / Q000379:methods*; D061905:Single-Domain Antibodies / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D046911:Macromolecular Substances; D028861:Nuclear Pore Complex Proteins; D061905:Single-Domain Antibodies
keywords: E. coli; biochemistry; cell biology; epitope mapping; label displacement; nanobody; native purification; nuclear pore complex; site-specific; xenopus