title: Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.
abstract: Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.
authors.affiliation: Interfaculty Institute of Biochemistry, Eberhard-Karls University Tuebingen, Germany.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Braun
authors.affiliation: Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany.
authors.forename: Bjoern
authors.initials: B
authors.lastname: Traenkle
authors.affiliation: Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany.
authors.forename: Philipp A
authors.initials: PA
authors.lastname: Koch
authors.affiliation: Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Emele
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany.
authors.forename: Frederik
authors.initials: F
authors.lastname: Weiss
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Poetz
authors.affiliation: Interfaculty Institute of Biochemistry, Eberhard-Karls University Tuebingen, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Stehle
authors.affiliation: Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany.|Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
mesh_terms: D000915:Antibody Affinity; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D008853:Microscopy; D008958:Models, Molecular; D009154:Mutation; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D040901:Proteomics / Q000379:methods*; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D000939:Epitopes; D019151:Peptide Library; D010455:Peptides; D061905:Single-Domain Antibodies