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Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy

Nature Nanotechnology volume 11, pages 89–94 (2016)Cite this article

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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.

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Figure 1: Model of monoST based on the crystal structures of SA and ST.
Figure 2: ITC measurements of ST constructs and SII peptide.
Figure 3: Characterization of SII:monoST as a general handhold system in AFM-based SMFS.
Figure 4: Comparison of SII:monoST unbinding forces depending on the placement of SII on the termini of the probed protein.

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Acknowledgements

This work was supported by the European Research Council (Cellufuel, Advanced Grant 294438) and the German Research Foundation (SFB1032-A01). The authors thank M. Gautel for providing the titin kinase construct, IBA for providing unmodified Strep-Tactin, M.A. Jobst for AFM software implementation, W. Ott for discussions, S.W. Stahl and A. Zeder for initial tests with Strep-Tactin in AFM force spectroscopy, K. Erlich for proof reading and A. Kardinal and T. Nicolaus for laboratory support.

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Authors and Affiliations

  1. Center for Nanoscience and Department of Physics, Ludwig Maximilians University of Munich, Amalienstraße 54, Munich, 80799, Germany

    Fabian Baumann, Magnus S. Bauer, Lukas F. Milles, Hermann E. Gaub & Diana A. Pippig

  2. Randall Division of Cell and Molecular Biophysics and Cardiovascular Division, New Hunt's House, King's College London, London, SE1 1UL, UK

    Alexander Alexandrovich

  3. Center for Integrated Protein Science Munich, Ludwig Maximilians University of Munich, Butenandtstraße 5–13, Munich, 81377, Germany

    Diana A. Pippig

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Contributions

H.E.G. and D.A.P. conceived the idea and designed the experiments. Experiments were carried out and evaluated by F.B., M.S.B. and D.A.P. L.F.M. provided force spectroscopy evaluation software and advice. A.A. prepared TK. D.A.P. wrote the manuscript with input from all authors.

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Correspondence to Diana A. Pippig.

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Baumann, F., Bauer, M., Milles, L. et al. Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy. Nature Nanotech 11, 89–94 (2016). https://doi.org/10.1038/nnano.2015.231

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