scholarly journals Ripping RNA by Force using Gaussian Network Models

2016 ◽  
Author(s):  
Changbong Hyeon ◽  
D. Thirumalai
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Cross Correlation ◽  
Network Models ◽  
Force Extension ◽  
Correlation Matrices ◽  
Rna Molecules ◽  
Allosteric Communication ◽  
Ion Effects ◽  
Gaussian Network

AbstractUsing force as a probe to map the folding landscapes of RNA molecules has become a reality thanks to major advances in single molecule pulling experiments. Although the unfolding pathways under tension are complicated to predict studies in the context of proteins have shown that topology plays is the major determinant of the unfolding landscapes. By building on this finding we study the responses of RNA molecules to force by adapting Gaussian network model (GNM) that represents RNAs using a bead-spring network with isotropic interactions. Cross-correlation matrices of residue fluctuations, which are analytically calculated using GNM even upon application of mechanical force, show distinct allosteric communication as RNAs rupture. The model is used to calculate the force-extension curves at full thermodynamic equilibrium, and the corresponding unfolding pathways of four RNA molecules subject to a quasi-statically increased force. Our study finds that the analysis using GNM captures qualitatively the unfolding pathway of T. ribozyme elucidated by the optical tweezers measurement. However, the simple model is not sufficient to capture subtle features, such as bifurcation in the unfolding pathways or the ion effects, in the forced-unfolding of RNAs.

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.

Unfolding Single RNA Molecules with Optical Tweezers

2001 ◽  
Vol 7 (S2) ◽  
pp. 26-27
Author(s):  
Carlos Bustamante ◽  
Jan Liphardt ◽  
Bibiana Onoa ◽  
Steven B. Smith ◽  
Delphine Collin ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Structural Features ◽  
Rna Molecules ◽  
Group I ◽  
Tertiary Folding ◽  
Tertiary Interactions

RNA molecules must fold into specific three-dimensional shapes to perform their structural and catalytic functions. Unlike proteins, RNAs secondary structural features are usually stable enough to form by themselves in solution. The reason is that in RNA, the stabilization energy gained from the formation of secondary structure is substantially larger than the energies involved in tertiary interactions. As a result, the formation of tertiary interactions is expected to alter only slightly the pre-existing secondary structural contacts. Moreover, secondary structure prediction is robust and can be made without taking into consideration tertiary folding. However, bulk studies of the energetics and kinetics of their secondary and tertiary folding are often frustrated by the presence of multiple species and multiple folding pathways in solution. These problems are circumvented in single-molecule studies in which the folding/unfolding trajectories of the individual molecules can be followed. The T. thermophila group I intron ribozyme is organized into several domains whose mechanical unfolding can be investigated independently, and whose tertiary contacts are stabilized by numerous Mg++ ions.We have begun characterization of the ribozyme by analysis of the P5abc domain because:

scholarly journals Elucidation of ion effects on the Thermodynamics of RNA Folding

2018 ◽  
Author(s):  
Natalia A. Denesyuk ◽  
D. Thirumalai
Keyword(s):  
Free Energy ◽  
Single Molecule ◽  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Rna Folding ◽  
Coarse Grained ◽  
Significant Advance ◽  
Rna Molecules ◽  
Ion Effects ◽  
The Difference

AbstractHow ions affect RNA folding thermodynamics and kinetics is an important but a vexing problem that remains unsolved. Experiments have shown that the free energy change, ΔG(c), of RNA upon folding varies with the salt concentration (c) as, ΔG(c) = kc ln c + const, where the coefficient kc is proportional to the difference in the uptake of ions (ion preferential coefficient), ΔΓ, between the folded and unfolded states. We performed simulations of a coarse-grained model, by modeling electrostatic interactions implicitly and with explicit representation of ions, to elucidate the molecular underpinnings of the relationship between folding free energy and ion preferential coefficient. Without any input from experiments, the simulations quantitatively reproduce the heat capacity for the −1 frame shifting pseudoknot (PK) from Beet Western Yellow Virus, thus validating the model. We show that ΔG(c) calculated directly from ΔΓ varies linearly with ln c (c < 0.2M), for a hairpin and the PK, thus demonstrating a molecular link between the two quantities for RNA molecules that undergo substantial conformational changes during folding. Explicit ion simulations also show the linear dependence of ΔG(c) on ln c at all c with kc = 2kBT, except that ΔG(c) values are shifted by about 2 kcal/mol higher than experiments at all salt concentrations. The discrepancy is due to an underestimate the Γ values for both the folded and unfolded states, while giving accurate values for ΔΓ. The predictions for the salt dependence of ΔΓ are amenable to test using single molecule pulling experiments. Our simulations, representing a significant advance in quantitatively describing ion effects in RNA, show that the framework provided here can be used to obtain accurate thermodynamics of RNA folding.

scholarly journals Optical tweezers in single-molecule biophysics

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Carlos J. Bustamante ◽  
Yann R. Chemla ◽  
Shixin Liu ◽  
Michelle D. Wang
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Single Molecule Biophysics

scholarly journals Single-molecule spectroelectrochemical cross-correlation during redox cycling in recessed dual ring electrode zero-mode waveguides

2017 ◽  
Vol 8 (8) ◽  
pp. 5345-5355 ◽  
Author(s):  
Donghoon Han ◽  
Garrison M. Crouch ◽  
Kaiyu Fu ◽  
Lawrence P. Zaino III ◽  
Paul W Bohn
Keyword(s):  
Electron Transfer ◽  
Single Molecule ◽  
Cross Correlation ◽  
Zero Mode ◽  
Redox Cycling ◽  
Ring Electrode ◽  
Dual Ring

The ability of zero-mode waveguides (ZMW) to guide light into subwavelength-diameter nanoapertures has been exploited for studying electron transfer dynamics in zeptoliter-volume nanopores under single-molecule occupancy conditions.

Molecular Motors: Force and Movement Generated by Single Myosin II Molecules

Physiology ◽  
2002 ◽  
Vol 17 (5) ◽  
pp. 213-218 ◽  
Author(s):  
Caspar Rüegg ◽  
Claudia Veigel ◽  
Justin E. Molloy ◽  
Stephan Schmitz ◽  
John C. Sparrow ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Molecular Motors ◽  
Molecular Motor ◽  
Myosin Ii ◽  
Force Production ◽  
Myosin Isoforms ◽  
Single Molecule Level ◽  
Recent Developments ◽  
Muscle Myosin

Muscle myosin II is an ATP-driven, actin-based molecular motor. Recent developments in optical tweezers technology have made it possible to study movement and force production on the single-molecule level and to find out how different myosin isoforms may have adapted to their specific physiological roles.

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.

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