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

TY - JOUR

T1 - Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy

AU - Baumann, Fabian

AU - Bauer, Magnus S.

AU - Milles, Lukas F.

AU - Alexandrovich, Alexander

AU - Gaub, Hermann E.

AU - Pippig, Diana A.

PY - 2016/1/1

Y1 - 2016/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84954367852&partnerID=8YFLogxK

U2 - 10.1038/nnano.2015.231

DO - 10.1038/nnano.2015.231

M3 - Article

AN - SCOPUS:84954367852

SN - 1748-3387

VL - 11

SP - 89

EP - 94

JO - Nature Nanotechnology

JF - Nature Nanotechnology

IS - 1

ER -