Nanomechanics of Streptavidin Hubs for Molecular Materials

Kim, Minkyu; Wang, Chien Chung; Benedetti, Fabrizio; Rabbi, Mahir; Bennett, Vann; Marszałek, Piotr E.

doi:10.1002/adma.201103316

Cited by 27 publications

(61 citation statements)

References 55 publications

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“…This is in agreement with former SMFS studies on the disruption of the SA dimer interface, which was found to occur at ∼100 pN (ref. 37). If high forces need to be probed, as in our exemplary GFP-unfolding experiment, monoST is a superior choice to conventional tetraST.…”

Section: Dynamic Smfs Of the Sii:monost Interactionmentioning

confidence: 99%

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

Baumann¹,

Bauer²,

Milles³

et al. 2015

Nature Nanotech

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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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Section: Dynamic Smfs Of the Sii:monost Interactionmentioning

confidence: 99%

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

Baumann¹,

Bauer²,

Milles³

et al. 2015

Nature Nanotech

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“…We hypothesize that high rupture forces, < F rupture-high >, are measured when Strep -tag ligands are removed from monomers belonging to one Strep -Tactin dimer and low rupture forces, < F rupture-low >, are measured when Strep -tag ligands are bound to two different dimers within the same Strep -Tactin tetramer (For details, see Supporting Information). Based on our recent study of streptavidin tetramers, [21] it is also possible that some of the rupture forces measured here may actually represent the forced separation of Strep -Tactin dimers from each other and not unbinding Strep -tag ligands. This hypothesis will be further tested by connecting our force probes directly to Strep -Tactin monomers (at the DNA level) and measuring the mechanical strength of the association of Strep -Tactin monomers within tetramers.…”

mentioning

confidence: 97%

“…When probing molecular complexes with higher affinities that will likely rupture at greater forces exceeding the unfolding force of I27, the presence of both mechanically weak and strong reference modules will be advantageous. [21] …”

mentioning

confidence: 99%

A Nanoscale Force Probe for Gauging Intermolecular Interactions

et al. 2012

Self Cite

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“…Indeed, the structure of streptavidin can be disrupted thermally, (62) or by mechanical stress. (27) An atomic force microscope (AFM) applying a stretching force of 100 pN at the pulling speed of 500 nm/s consistently ruptures the 4 hydrogen bonds binding the two dimers of streptavidin together. (63) Similarly, a recent theoretical study modeled the effects of a nanopore's electric field on streptavidin and found potential changes in streptavidin's secondary structure under certain pulling conditions at 2 V through a 10-nm diameter hour-glass-shaped nanopore in 1 M KCl.…”

Section: Monovalent Streptavidinmentioning

confidence: 99%

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

Carlsen

Tabard‐Cossa

2020

Preprint

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Solid-state nanopores have been used extensively in biomolecular studies involving DNA and proteins. However, the interpretation of signals generated by the translocation of proteins or protein-DNA complexes remains challenging. Here, we investigate the behavior of monovalent streptavidin and the complex it forms with short biotinylated DNA over a range of nanopore sizes, salts and voltages. We describe a simple geometric model that is broadly applicable and employ it to explain observed variations in conductance blockage and dwell time with experimental conditions. The general approach developed here underscores the value of nanopore-based protein analysis and represents progress toward the interpretation of complex translocation signals. STATEMENT OF SIGNIFICANCENanopore sensing allows investigation of biomolecular structure in aqueous solution, including electricfield-induced changes in protein conformation. This nanopore-based study probes the tetramer-dimer transition of streptavidin, observing the effects of increasing voltage with varying salt type and concentration. Binding of biotinylated DNA to streptavidin boosts the complex's structural integrity, while complicating signal analysis. We describe a broadly applicable geometric approach that maps stepwise changes in the nanopore signal to real-time conformational transitions. These results represent important progress toward accurate interpretation of nanopore signals generated by macromolecular complexes.

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Nanomechanics of Streptavidin Hubs for Molecular Materials

Cited by 27 publications

References 55 publications

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

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

A Nanoscale Force Probe for Gauging Intermolecular Interactions

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

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