An Optical Trap Combined with Three-Color FRET

Lee, Sanghwa; Hohng, Sungchul

doi:10.1021/ja408767p

Cited by 30 publications

(27 citation statements)

References 32 publications

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“…But true simultaneous measurement along more than one reaction coordinate could provide still greater insight, helping to address such issues as secondary vs. tertiary structure formation, coupling between the folding of different structural elements, and discrimination between ligand-bound and ligand-free states [59, 80, 86, 103]. New methodologies to record more than one reaction coordinate, which are being vigorously pursued but are still in their infancy, include combined OT and smFRET (“Force-FRET”) [89, 127–130], multicolor smFRET [87, 88] (which can also be combined with OT [131]), and smFRET combined with AFM [132, 133] or magnetic tweezers [134]. Each single-molecule approach individually poses its own unique set of technical challenges, and these tend to get compounded when techniques are combined.…”

Section: Discussionmentioning

confidence: 99%

Single-molecule studies of riboswitch folding

Savinov

Perez

Block

2014

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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The folding dynamics of riboswitches are central to their ability to modulate gene expression in response to environmental cues. In most cases, a structural competition between the formation of a ligand-binding aptamer and an expression platform (or some other competing off-state) determines the regulatory outcome. Here, we review single-molecule studies of riboswitch folding and function, predominantly carried out using single-molecule FRET or optical trapping approaches. Recent results have supplied new insights into riboswitch folding energy landscapes, the mechanisms of ligand binding, the roles played by divalent ions, the applicability of hierarchical folding models, and kinetic vs. thermodynamic control schemes. We anticipate that future work, based on improved data sets and potentially combining multiple experimental techniques, will enable the development of more complete models for complex RNA folding processes.

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Section: Discussionmentioning

confidence: 99%

Single-molecule studies of riboswitch folding

Savinov

Perez

Block

2014

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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“…The singly labeled DNA fragment used to examine the nucleosome remodeling activity of CSB was generated using a primer end-labeled with Cy5 at the 5΄ end and a second primer containing biotin at the 5΄ end. Mononucleosomes containing Cy3-labeled H2A were prepared as described (21). Mononucleosomes were reconstituted using salt gradient dialysis (21).…”

Section: Methodsmentioning

confidence: 99%

NAP1L1 accelerates activation and decreases pausing to enhance nucleosome remodeling by CSB

Lee

Lake

Kirk

et al. 2017

Nucleic Acids Research

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Cockayne syndrome protein B (CSB) belongs to the SWI2/SNF2 ATP-dependent chromatin remodeler family, and CSB is the only ATP-dependent chromatin remodeler essential for transcription-coupled nucleotide excision DNA repair. CSB alone remodels nucleosomes ∼10-fold slower than the ACF remodeling complex. Strikingly, NAP1-like histone chaperones interact with CSB and greatly enhance CSB-mediated chromatin remodeling. While chromatin remodeling by CSB and NAP1-like proteins is crucial for efficient transcription-coupled DNA repair, the mechanism by which NAP1-like proteins enhance chromatin remodeling by CSB remains unknown. Here we studied CSB's DNA-binding and nucleosome-remodeling activities at the single molecule level in real time. We also determined how the NAP1L1 chaperone modulates these activities. We found that CSB interacts with DNA in two principle ways: by simple binding and a more complex association that involves gross DNA distortion. Remarkably, NAP1L1 suppresses both these interactions. Additionally, we demonstrate that nucleosome remodeling by CSB consists of three distinct phases: activation, translocation and pausing, similar to ACF. Importantly, we found that NAP1L1 promotes CSB-mediated remodeling by accelerating both activation and translocation. Additionally, NAP1L1 increases CSB processivity by decreasing the pausing probability during translocation. Our study, therefore, uncovers the different steps of CSB-mediated chromatin remodeling that can be regulated by NAP1L1.

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“…Presently, smFRET coupled with optical tweezers, magnetic tweezers, or AFM has shown very promising results. 58,128,142 Potential future directions could involve the combination of smFRET with biomembrane force probe (BFP) 131 and lattice light-sheet microscopy. 133 Also, resolution of smFRET efficiency in 3D orientation will also be a major future advancement.…”

Section: Discussionmentioning

confidence: 99%

“…Although force-coupled smFRET using optical tweezers has been used to study kinesin stepping, DNA hairpin unfolding, and DNA-protein interaction, 58,130 there is a major problem caused by the high photon flux of a trapping laser, which can bleach fluorophores as well as kill live cells. For force coupled smFRET using AFM, one major challenge is the high inherent cantilever stiffness and incoherent reflection from cantilever.…”

Section: Applications Of Smfretmentioning

confidence: 99%

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Single-molecule fluorescence resonance energy transfer in molecular biology

et al. 2016

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Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique for studying the conformation dynamics and interactions of individual biomolecules. In this review, we describe the concept and principle of smFRET, illustrate general instrumentation and microscopy settings for experiments, and discuss the methods and algorithms for data analysis. Subsequently, we review applications of smFRET in protein conformational changes, ion channel open-close properties, receptor-ligand interactions, nucleic acid structure regulation, vesicle fusion, and force induced conformational dynamics. Finally, we discuss the main limitations of smFRET in molecular biology.

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An Optical Trap Combined with Three-Color FRET

Cited by 30 publications

References 32 publications

Single-molecule studies of riboswitch folding

Single-molecule studies of riboswitch folding

NAP1L1 accelerates activation and decreases pausing to enhance nucleosome remodeling by CSB

Single-molecule fluorescence resonance energy transfer in molecular biology

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