Dissecting the Conformational Dynamics-Modulated Enzyme Catalysis with Single-Molecule FRET

2018 ◽  
Vol 122 (23) ◽  
pp. 6179-6187 ◽  
Author(s):  
Shaowen Wu ◽  
Jianwei Liu ◽  
Wenning Wang
Keyword(s):  
Single Molecule ◽  
Enzyme Catalysis ◽  
Conformational Dynamics ◽  
Single Molecule Fret

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 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 Single Molecule FRET Studies of Protein Conformational Landscapes: 3 Prototypic Examples for the Relation Between Conformational Dynamics and Function

2011 ◽  
Vol 100 (3) ◽  
pp. 474a-475a
Author(s):  
Markus Richert ◽  
Dymitro Rodnin ◽  
Carola S. Hengstenberg ◽  
Thomas Peulen ◽  
Alessandro Valeri ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Single Molecule Fret ◽  
Dynamics And Function ◽  
And Function

scholarly journals 631: Single-molecule FRET reveals CFTR conformational dynamics during channel gating

2021 ◽  
Vol 20 ◽  
pp. S299-S300
Author(s):  
P. Liyanage ◽  
K. Mun ◽  
S. Yarlagadda ◽  
Y. Huang ◽  
A. Naren
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Channel Gating ◽  
Single Molecule Fret

scholarly journals Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET

2017 ◽  
Author(s):  
Mengyi Yang ◽  
Sijia Peng ◽  
Ruirui Sun ◽  
Jingdi Lin ◽  
Nan Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nuclease Activity ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fret ◽  
Allosteric Communication ◽  
Target Binding

SummaryOff-target binding and cleavage by Cas9 pose as major challenges in its applications. How conformational dynamics of Cas9 governs its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms all spontaneously transits between three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We furthermore uncovered a surprising long-range allosteric communication between the HNH domain and RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.

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 Linking Enzyme Conformational Dynamics To Catalytic Function With Single-molecule FRET

2009 ◽  
Vol 96 (3) ◽  
pp. 73a-74a
Author(s):  
Yan-Wen Tan ◽  
Jeffrey A. Hanson ◽  
Jason Brokaw ◽  
Jhih-Wei Chu ◽  
Haw Yang
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Single Molecule Fret ◽  
Catalytic Function

scholarly journals Ultrafast Brownian-ratchet mechanism for protein translocation by a AAA+ machine

2020 ◽  
Author(s):  
Hisham Mazal ◽  
Marija Iljina ◽  
Inbal Riven ◽  
Gilad Haran
Keyword(s):  
Single Molecule ◽  
Protein Translocation ◽  
Conformational Dynamics ◽  
Brownian Ratchet ◽  
Time Dynamics ◽  
Single Molecule Fret ◽  
Differential Behavior ◽  
Amplitude Fluctuations ◽  
Translocation Mechanism ◽  
Substrate Proteins

AbstractAAA+ ring-shaped machines, such as ClpB and Hsp104, mediate substrate translocation through their central channel by a set of pore loops. Recent structural studies suggested a universal hand-over-hand translocation mechanism, in which pore loops are moving rigidly in tandem with their corresponding subunits. However, functional and biophysical studies are in discord with this model. Here, we directly measure the real-time dynamics of the pore loops of ClpB and their response to substrate binding, using single-molecule FRET spectroscopy. All pore loops undergo large-amplitude fluctuations on the microsecond timescale, and change their conformation upon interaction with substrate proteins. Pore-loop conformational dynamics are modulated by nucleotides and strongly correlate with disaggregation activity. The differential behavior of the pore loops along the axial channel points to a fast Brownian-ratchet translocation mechanism, which likely acts in parallel to the much slower hand-over-hand process.

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