title: Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy.
abstract: Strep-Tactin, an engineered form of streptavidin, binds avidly to the genetically encoded peptide Strep-tag II in a manner comparable to streptavidin binding to biotin. These interactions have been used in protein purification and detection applications. However, in single-molecule studies, for example using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), the tetravalency of these systems impedes the measurement of monodispersed data. Here, we introduce a monovalent form of Strep-Tactin that harbours a unique binding site for Strep-tag II and a single cysteine that allows Strep-Tactin to specifically attach to the atomic force microscope cantilever and form a consistent pulling geometry to obtain homogeneous rupture data. Using AFM-SMFS, the mechanical properties of the interaction between Strep-tag II and monovalent Strep-Tactin were characterized. Rupture forces comparable to biotin:streptavidin unbinding were observed. Using titin kinase and green fluorescent protein, we show that monovalent Strep-Tactin is generally applicable to protein unfolding experiments. We expect monovalent Strep-Tactin to be a reliable anchoring tool for a range of single-molecule studies.
authors.affiliation: Center for Nanoscience and Department of Physics, Ludwig Maximilians University of Munich, Amalienstraße 54, Munich 80799, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Baumann
authors.affiliation: Center for Nanoscience and Department of Physics, Ludwig Maximilians University of Munich, Amalienstraße 54, Munich 80799, Germany.
authors.forename: Magnus S
authors.initials: MS
authors.lastname: Bauer
authors.affiliation: Center for Nanoscience and Department of Physics, Ludwig Maximilians University of Munich, Amalienstraße 54, Munich 80799, Germany.
authors.forename: Lukas F
authors.initials: LF
authors.lastname: Milles
authors.affiliation: Randall Division of Cell and Molecular Biophysics and Cardiovascular Division, New Hunt's House, King's College London, London SE1 1UL, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Alexandrovich
authors.affiliation: Center for Nanoscience and Department of Physics, Ludwig Maximilians University of Munich, Amalienstraße 54, Munich 80799, Germany.
authors.forename: Hermann E
authors.initials: HE
authors.lastname: Gaub
authors.affiliation: Center for Nanoscience and Department of Physics, Ludwig Maximilians University of Munich, Amalienstraße 54, Munich 80799, Germany.|Center for Integrated Protein Science Munich, Ludwig Maximilians University of Munich, Butenandtstraße 5-13, Munich 81377, Germany.
authors.forename: Diana A
authors.identifier: http://orcid.org/0000-0001-7472-7999
authors.initials: DA
authors.lastname: Pippig
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D018625:Microscopy, Atomic Force / Q000379:methods*; D015336:Molecular Probe Techniques; D009842:Oligopeptides / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure
chemical_list: C099517:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; D009842:Oligopeptides; D019809:Streptavidin