Direct observation of the external force mediated conformational dynamics of an IHF bound Holliday junction

2018 ◽  
Vol 207 ◽  
pp. 251-265
Author(s):  
Subhas C. Bera ◽  
Tapas Paul ◽  
A. N. Sekar Iyengar ◽  
Padmaja P. Mishra
Keyword(s):  
Dna Binding ◽  
External Force ◽  
Single Molecule ◽  
Energy Landscape ◽  
Conformational Dynamics ◽  
Dna Binding Protein ◽  
Integration Host Factor ◽  
Holliday Junctions ◽  
Holliday Junction ◽  
Single Molecule Fret

We have investigated the isomerization dynamics and plausible energy landscape of 4-way Holliday junctions (4WHJs) bound to integration host factor (IHF, a DNA binding protein), considering the effect of applied external force, by single-molecule FRET methods.

scholarly journals Slow, heterogeneous NFκB single molecule conformational dynamics and implications for function

2021 ◽  
Author(s):  
Wei Chen ◽  
Wei Liu ◽  
Peter Wolynes ◽  
Elizabeth A. Komives
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Electrostatic Interactions ◽  
Bound States ◽  
Conformational Dynamics ◽  
Dna Binding Domains ◽  
Single Molecule Fret ◽  
Binding Domains ◽  
Lower Amplitude ◽  
Slow Motions

The transcription factor NFκB (RelA-p50) is a multidomain protein that binds DNA and its inhibitor, IκBα with apparently different conformations. We used single-molecule FRET to characterize the interdomain motions of the N-terminal DNA-binding domains in the free protein and also in various bound states. Several surprising results emerged from this study. First, the domains moved with respect to each other on several widely different timescales from hundreds of milliseconds to minutes. The free NFκB displayed stochastic motions leading to a broad distribution of states, ranging from very low-FRET states to high-FRET states. Varying the ionic strength altered the slow motions suggesting that they may be due to different weak electrostatic interactions between the domains creating a rugged energy landscape. Third, the DNA-binding domains continued to be mobile even when the protein was bound to its cognate DNA, but in this case the majority of the states were either high-FRET, a state expected from the available x-ray structures, or low-FRET, a state consistent with one of the DNA-binding domains dissociated. The fluctuations of the DNA-bound states were of lower amplitude and slightly faster frequency. Fourth, the inhibitor, IκBα freezes the domains into a low-FRET state, expected to be incapable of binding DNA. Neutralization of five acidic residues in the IκBα PEST sequence, which was previously shown to impair IκBαs ability to strip NFκB from the DNA, also impaired its ability to freeze the domains into a low-FRET state indicating that the freezing of motions of the DNA-binding domains is essential for efficient molecular stripping.

Conformational Dynamics of DNA Hairpins at Millisecond Resolution Obtained from Analysis of Single-Molecule FRET Histograms

2013 ◽  
Vol 117 (50) ◽  
pp. 16105-16109 ◽  
Author(s):  
Roman Tsukanov ◽  
Toma E. Tomov ◽  
Yaron Berger ◽  
Miran Liber ◽  
Eyal Nir
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Dna Hairpins ◽  
Single Molecule Fret

scholarly journals Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein

2018 ◽  
Vol 46 (21) ◽  
pp. 11287-11302 ◽  
Author(s):  
Parminder Kaur ◽  
Matthew J Longley ◽  
Hai Pan ◽  
Hong Wang ◽  
William C Copeland
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Single Stranded Dna ◽  
Dna Wrapping

scholarly journals Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

2018 ◽  
Vol 37 (21) ◽  
Author(s):  
Florence Husada ◽  
Kiran Bountra ◽  
Konstantinos Tassis ◽  
Marijn Boer ◽  
Maria Romano ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Single Molecule Fret

scholarly journals Slow domain reconfiguration causes power-law kinetics in a two-state enzyme

2018 ◽  
Vol 115 (3) ◽  
pp. 513-518 ◽  
Author(s):  
Iris Grossman-Haham ◽  
Gabriel Rosenblum ◽  
Trishool Namani ◽  
Hagen Hofmann
Keyword(s):  
Free Energy ◽  
Single Molecule ◽  
Power Law ◽  
Energy Landscape ◽  
Rate Equations ◽  
Free Energy Landscape ◽  
Closed Domain ◽  
Single Molecule Fret ◽  
Power Law Kinetics ◽  
Interdomain Interactions

Protein dynamics are typically captured well by rate equations that predict exponential decays for two-state reactions. Here, we describe a remarkable exception. The electron-transfer enzyme quiescin sulfhydryl oxidase (QSOX), a natural fusion of two functionally distinct domains, switches between open- and closed-domain arrangements with apparent power-law kinetics. Using single-molecule FRET experiments on time scales from nanoseconds to milliseconds, we show that the unusual open-close kinetics results from slow sampling of an ensemble of disordered domain orientations. While substrate accelerates the kinetics, thus suggesting a substrate-induced switch to an alternative free energy landscape of the enzyme, the power-law behavior is also preserved upon electron load. Our results show that the slow sampling of open conformers is caused by a variety of interdomain interactions that imply a rugged free energy landscape, thus providing a generic mechanism for dynamic disorder in multidomain enzymes.

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