Discriminating small molecule DNA binding modes by single molecule force spectroscopy

AbstractThe small molecule biotin and the homotetrameric protein streptavidin (SA) form a stable and robust complex that plays a pivotal role in many biotechnological and medical applications. In particular, the biotin-streptavidin linkage is frequently used in single molecule force spectroscopy (SMFS) experiments. Recent data suggest that biotin-streptavidin bonds show strong directional dependence and a broad range of multi-exponential lifetimes under load. Here, we investigate engineered SA variants with different valencies and a unique tethering point under constant forces using a magnetic tweezer assay. We observed two orders-of-magnitude differences in the lifetimes, which we attribute to the distinct force loading geometries in the different SA variants. We identified an especially long-lived tethering geometry that will facilitate ultra-stable SMFS experiments and pave the way for new biotechnological applications.

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Investigation of the binding modes between AIE-active molecules and dsDNA by single molecule force spectroscopy

Nanoscale ◽

10.1039/c5nr01247c ◽

2015 ◽

Vol 7 (19) ◽

pp. 8939-8945 ◽

Cited By ~ 21

Author(s):

Ying Chen ◽

Ke Ma ◽

Ting Hu ◽

Bo Jiang ◽

Bin Xu ◽

...

Keyword(s):

Single Molecule ◽

Force Spectroscopy ◽

Binding Modes ◽

Single Molecule Force Spectroscopy ◽

Aggregation Induced Emission ◽

Double Stranded Dna

The binding modes between double-stranded DNA (dsDNA) and typical AIE (aggregation-induced emission)-active molecules were investigated using AFM-based single molecule force spectroscopy.

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Single molecule force spectroscopy studies of DNA denaturation by T4 gene 32 protein

Spectroscopy An International Journal ◽

10.1155/2004/403203 ◽

2004 ◽

Vol 18 (2) ◽

pp. 203-211 ◽

Cited By ~ 16

Author(s):

Mark C. Williams ◽

Kiran Pant ◽

Ioulia Rouzina ◽

Richard L. Karpel

Keyword(s):

Dna Binding ◽

Optical Tweezers ◽

Single Molecule ◽

Protein Interactions ◽

Force Spectroscopy ◽

Melting Transition ◽

Dna Molecules ◽

Force Extension ◽

Single Molecule Force Spectroscopy ◽

Gene 32

Single molecule force spectroscopy is an emerging technique that can be used to measure the biophysical properties of single macromolecules such as nucleic acids and proteins. In particular, single DNA molecule stretching experiments are used to measure the elastic properties of these molecules and to induce structural transitions. We have demonstrated that double‒stranded DNA molecules undergo a force‒induced melting transition at high forces. Force–extension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review the evidence of DNA melting in these experiments and discuss the example of DNA force‒induced melting in the presence of the single‒stranded DNA binding protein T4 gene 32. We show that this force spectroscopy technique is a useful probe of DNA–protein interactions, which allows us to obtain binding rates and binding free energies for these interactions.

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Single-Molecule Force Spectroscopy Reveals Multiple Binding Modes between DOPA and Different Rutile Surfaces

ChemPhysChem ◽

10.1002/cphc.201600374 ◽

2016 ◽

Vol 18 (11) ◽

pp. 1466-1469 ◽

Cited By ~ 18

Author(s):

Yiran Li ◽

Huanyu Liu ◽

Tiankuo Wang ◽

Meng Qin ◽

Yi Cao ◽

...

Keyword(s):

Single Molecule ◽

Force Spectroscopy ◽

Binding Modes ◽

Single Molecule Force Spectroscopy ◽

Multiple Binding

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Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy

eLife ◽

10.7554/elife.10319 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 30

Author(s):

Markus A Jobst ◽

Lukas F Milles ◽

Constantin Schoeler ◽

Wolfgang Ott ◽

Daniel B Fried ◽

...

Keyword(s):

Single Molecule ◽

Molecular Conformation ◽

Bacterial Species ◽

Force Spectroscopy ◽

Binding Mode ◽

Site Directed Mutagenesis ◽

Binding Modes ◽

Single Molecule Force Spectroscopy ◽

Vast Number ◽

Knock Out

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicteddual binding modes across multiple bacterial species, our approach opens up newpossibilities for understanding assembly and catalytic properties of a broadrange of multi-enzyme complexes.

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