scholarly journals Calculating the force-dependent unbinding rate of biological macromolecular bonds from force-ramp optical trapping assays

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Apurba Paul ◽  
Joshua Alper
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Optical Tweezer ◽  
Stochastic Simulations ◽  
Cumulative Distribution ◽  
Cellular Functions ◽  
Single Molecule Force Spectroscopy ◽  
Protein Ligand Interactions ◽  
Ligand Interactions

AbstractThe non-covalent biological bonds that constitute protein–protein or protein–ligand interactions play crucial roles in many cellular functions, including mitosis, motility, and cell–cell adhesion. The effect of external force ($$F$$ F ) on the unbinding rate ($${k}_{\text{off}}\left(F\right)$$ k off F ) of macromolecular interactions is a crucial parameter to understanding the mechanisms behind these functions. Optical tweezer-based single-molecule force spectroscopy is frequently used to obtain quantitative force-dependent dissociation data on slip, catch, and ideal bonds. However, analyses of this data using dissociation time or dissociation force histograms often quantitatively compare bonds without fully characterizing their underlying biophysical properties. Additionally, the results of histogram-based analyses can depend on the rate at which force was applied during the experiment and the experiment’s sensitivity. Here, we present an analytically derived cumulative distribution function-like approach to analyzing force-dependent dissociation force spectroscopy data. We demonstrate the benefits and limitations of the technique using stochastic simulations of various bond types. We show that it can be used to obtain the detachment rate and force sensitivity of biological macromolecular bonds from force spectroscopy experiments by explicitly accounting for loading rate and noisy data. We also discuss the implications of our results on using optical tweezers to collect force-dependent dissociation data.

scholarly journals Calculating the force-dependent unbinding rate of biological macromolecular bonds from the force-ramp optical trapping assays

2021 ◽  
Author(s):  
Apurba Paul ◽  
Joshua Alper
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Optical Tweezer ◽  
Stochastic Simulations ◽  
Cumulative Distribution ◽  
Cellular Functions ◽  
Single Molecule Force Spectroscopy ◽  
Protein Ligand Interactions ◽  
Ligand Interactions

AbstractThe non-covalent biological bonds that constitute protein-protein or protein-ligand interactions play crucial roles in many cellular functions, including mitosis, motility, and cell-cell adhesion. The effect of external force (F) on the unbinding rate (koff(F)) of macromolecular interactions is a crucial parameter to understanding the mechanisms behind these functions. Optical tweezer-based single-molecule force spectroscopy is frequently used to obtain quantitative force-dependent dissociation data on slip, catch, and ideal bonds. However, analyses of this data using dissociation time or dissociation force histograms often quantitatively compare bonds without fully characterizing their underlying biophysical properties. Additionally, the results of histogram-based analyses can depend on the rate at which force was applied during the experiment and the experiment’s sensitivity. Here, we present an analytically derived cumulative distribution function-like approach to analyzing force-dependent dissociation force spectroscopy data. We demonstrate the benefits and limitations of the technique using stochastic simulations of various bond types. We show that it can be used to obtain the detachment rate and force sensitivity of biological macromolecular bonds from force spectroscopy experiments by explicitly accounting for loading rate and noisy data. We also discuss the implications of our results on using optical tweezers to collect force-dependent dissociation data.

scholarly journals Calculating the force-dependent unbinding rate of biological macromolecular bonds from force-ramp optical trapping assays

2021 ◽  
Author(s):  
Apurba Paul ◽  
Joshua Alper
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Optical Tweezer ◽  
Stochastic Simulations ◽  
Cumulative Distribution ◽  
Cellular Functions ◽  
Single Molecule Force Spectroscopy ◽  
Protein Ligand Interactions ◽  
Ligand Interactions

Abstract The non-covalent biological bonds that constitute protein-protein or protein-ligand interactions play crucial roles in many cellular functions, including mitosis, motility, and cell-cell adhesion. The effect of external force (𝐹) on the unbinding rate (𝑘off(𝐹)) of macromolecular interactions is a crucial parameter to understanding the mechanisms behind these functions. Optical tweezer-based single-molecule force spectroscopy is frequently used to obtain quantitative force-dependent dissociation data on slip, catch, and ideal bonds. However, analyses of this data using dissociation time or dissociation force histograms often quantitatively compare bonds without fully characterizing their underlying biophysical properties. Additionally, the results of histogram-based analyses can depend on the rate at which force was applied during the experiment and the experiment’s sensitivity. Here, we present an analytically derived cumulative distribution function-like approach to analyzing force-dependent dissociation force spectroscopy data. We demonstrate the benefits and limitations of the technique using stochastic simulations of various bond types. We show that it can be used to obtain the detachment rate and force sensitivity of biological macromolecular bonds from force spectroscopy experiments by explicitly accounting for loading rate and noisy data. We also discuss the implications of our results on using optical tweezers to collect force-dependent dissociation data.

scholarly journals Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor

2018 ◽  
Vol 115 (46) ◽  
pp. 11688-11693 ◽  
Author(s):  
Thomas Suren ◽  
Daniel Rutz ◽  
Patrick Mößmer ◽  
Ulrich Merkel ◽  
Johannes Buchner ◽  
...  
Keyword(s):  
Free Energy ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Mechanical Load ◽  
Force Spectroscopy ◽  
Folding Pathway ◽  
Hormone Binding ◽  
Single Molecule Force Spectroscopy

The glucocorticoid receptor (GR) is a prominent nuclear receptor linked to a variety of diseases and an important drug target. Binding of hormone to its ligand binding domain (GR-LBD) is the key activation step to induce signaling. This process is tightly regulated by the molecular chaperones Hsp70 and Hsp90 in vivo. Despite its importance, little is known about GR-LBD folding, the ligand binding pathway, or the requirement for chaperone regulation. In this study, we have used single-molecule force spectroscopy by optical tweezers to unravel the dynamics of the complete pathway of folding and hormone binding of GR-LBD. We identified a “lid” structure whose opening and closing is tightly coupled to hormone binding. This lid is located at the N terminus without direct contacts to the hormone. Under mechanical load, apo-GR-LBD folds stably and readily without the need of chaperones with a folding free energy of 41 kBT (24 kcal/mol). The folding pathway is largely independent of the presence of hormone. Hormone binds only in the last step and lid closure adds an additional 12 kBT of free energy, drastically increasing the affinity. However, mechanical double-jump experiments reveal that, at zero force, GR-LBD folding is severely hampered by misfolding, slowing it to less than 1·s−1. From the force dependence of the folding rates, we conclude that the misfolding occurs late in the folding pathway. These features are important cornerstones for understanding GR activation and its tight regulation by chaperones.

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

2004 ◽  
Vol 18 (2) ◽  
pp. 203-211 ◽  
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.

scholarly journals Bioconjugation Strategies for Connecting Proteins to DNA-Linkers for Single-Molecule Force-Based Experiments

Nanomaterials ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2424
Author(s):  
Lyan M. van der Sleen ◽  
Katarzyna M. Tych
Keyword(s):  
Mechanical Properties ◽  
Atomic Force Microscopy ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Short Range ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Atomic Force

The mechanical properties of proteins can be studied with single molecule force spectroscopy (SMFS) using optical tweezers, atomic force microscopy and magnetic tweezers. It is common to utilize a flexible linker between the protein and trapped probe to exclude short-range interactions in SMFS experiments. One of the most prevalent linkers is DNA due to its well-defined properties, although attachment strategies between the DNA linker and protein or probe may vary. We will therefore provide a general overview of the currently existing non-covalent and covalent bioconjugation strategies to site-specifically conjugate DNA-linkers to the protein of interest. In the search for a standardized conjugation strategy, considerations include their mechanical properties in the context of SMFS, feasibility of site-directed labeling, labeling efficiency, and costs.

Dynamic single-molecule force spectroscopy using optical tweezers and nanopores

2013 ◽  
Author(s):  
Nadanai Laohakunakorn ◽  
Oliver Otto ◽  
Sebastian Sturm ◽  
Klaus Kroy ◽  
Ulrich F. Keyser
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy

scholarly journals Characterization of G-Quadruplexes Folding/Unfolding Dynamics and Interactions with Proteins from Single-Molecule Force Spectroscopy

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1579
Author(s):  
Yuanlei Cheng ◽  
Yashuo Zhang ◽  
Huijuan You
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Multiple Structures ◽  
Regulation Functions ◽  
Nucleic Acid Structures

G-quadruplexes (G4s) are stable secondary nucleic acid structures that play crucial roles in many fundamental biological processes. The folding/unfolding dynamics of G4 structures are associated with the replication and transcription regulation functions of G4s. However, many DNA G4 sequences can adopt a variety of topologies and have complex folding/unfolding dynamics. Determining the dynamics of G4s and their regulation by proteins remains challenging due to the coexistence of multiple structures in a heterogeneous sample. Here, in this mini-review, we introduce the application of single-molecule force–spectroscopy methods, such as magnetic tweezers, optical tweezers, and atomic force microscopy, to characterize the polymorphism and folding/unfolding dynamics of G4s. We also briefly introduce recent studies using single-molecule force spectroscopy to study the molecular mechanisms of G4-interacting proteins.

scholarly journals The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates

2021 ◽  
Author(s):  
Keith J. Mickolajczyk ◽  
Paul Dominic B. Olinares ◽  
Brian T. Chait ◽  
Shixin Liu ◽  
Tarun M. Kapoor
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Ribosome Biogenesis ◽  
Bound States ◽  
Weakly Bound ◽  
Aaa Atpase ◽  
Protein Ligand Interactions ◽  
Single Protein ◽  
Ligand Interactions ◽  
Catch Bonds

ABSTRACTCatch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential AAA (ATPase associated with diverse cellular activities) mechanoenzyme Mdn1 drives two separate steps in ribosome biogenesis, using its MIDAS domain to extract the ubiquitin-like (UBL) domain-containing proteins Rsa4 and Ytm1 from ribosomal precursors. However, it must subsequently release these assembly factors to reinitiate the enzymatic cycle. The mechanism underlying MIDAS-UBL switching between strongly- and weakly-bound states is unknown. Here, we use single-molecule optical tweezers to investigate the force-dependence of MIDAS-UBL binding. Parallel experiments with Rsa4 and Ytm1 show that forces up to ~4 pN, matching the magnitude of force produced by AAA proteins similar to Mdn1, enhance the MIDAS domain binding lifetime up to tenfold, and higher forces accelerate dissociation. Together, our studies indicate that Mdn1’s MIDAS domain forms catch bonds with more than one UBL-substrate, and provide insights into how mechanoregulation may contribute to the Mdn1 enzymatic cycle during ribosome biogenesis.

scholarly journals Mechanically tightening, untying and retying a protein trefoil knot by single-molecule force spectroscopy

2020 ◽  
Vol 11 (46) ◽  
pp. 12512-12521
Author(s):  
Han Wang ◽  
Hongbin Li
Keyword(s):  
Conformational Change ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy ◽  
Trefoil Knot ◽  
Knotted Protein

Optical tweezers are used to stretch a knotted protein along different directions to probe its unfolding–folding behaviors, and the conformational change of its knot structure.

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