scholarly journals Direct observation of ultrafast large-scale dynamics of an enzyme under turnover conditions

2018 ◽  
Vol 115 (13) ◽  
pp. 3243-3248 ◽  
Author(s):  
Haim Yuval Aviram ◽  
Menahem Pirchi ◽  
Hisham Mazal ◽  
Yoav Barak ◽  
Inbal Riven ◽  
...  
Keyword(s):  
Single Molecule ◽  
Active Site ◽  
Adenylate Kinase ◽  
Large Scale ◽  
Turnover Rate ◽  
Conformational Dynamics ◽  
Mutual Orientation ◽  
Single Molecule Fret ◽  
Domain Motions ◽  
Active Site Mutants

The functional cycle of many proteins involves large-scale motions of domains and subunits. The relation between conformational dynamics and the chemical steps of enzymes remains under debate. Here we show that in the presence of substrates, domain motions of an enzyme can take place on the microsecond time scale, yet exert influence on the much-slower chemical step. We study the domain closure reaction of the enzyme adenylate kinase from Escherichia coli while in action (i.e., under turnover conditions), using single-molecule FRET spectroscopy. We find that substrate binding increases dramatically domain closing and opening times, making them as short as ∼15 and ∼45 µs, respectively. These large-scale conformational dynamics are likely the fastest measured to date, and are ∼100–200 times faster than the enzymatic turnover rate. Some active-site mutants are shown to fully or partially prevent the substrate-induced increase in domain closure times, while at the same time they also reduce enzymatic activity, establishing a clear connection between the two phenomena, despite their disparate time scales. Based on these surprising observations, we propose a paradigm for the mode of action of enzymes, in which numerous cycles of conformational rearrangement are required to find a mutual orientation of substrates that is optimal for the chemical reaction.

scholarly journals Aminoglycoside interactions and impacts on the eukaryotic ribosome

2017 ◽  
Vol 114 (51) ◽  
pp. E10899-E10908 ◽  
Author(s):  
Irina Prokhorova ◽  
Roger B. Altman ◽  
Muminjon Djumagulov ◽  
Jaya P. Shrestha ◽  
Alexandre Urzhumtsev ◽  
...  
Keyword(s):  
Single Molecule ◽  
Large Scale ◽  
Conformational Dynamics ◽  
Therapeutic Interventions ◽  
80S Ribosome ◽  
Eukaryotic Translation ◽  
Eukaryotic Ribosome ◽  
X Ray Crystallography ◽  
Single Molecule Fret ◽  
Translation Mechanism

Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-Å resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6′-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding.

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 Conformational dynamics modulate the catalytic activity of the molecular chaperone Hsp90

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sophie L. Mader ◽  
Abraham Lopez ◽  
Jannis Lawatscheck ◽  
Qi Luo ◽  
Daniel A. Rutz ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Active Site ◽  
Large Scale ◽  
Atp Hydrolysis ◽  
Mechanistic Model ◽  
Ion Pairs ◽  
Conformational Dynamics ◽  
Heat Shock Protein 90 ◽  
Eukaryotic Cell ◽  
Atpase Reaction

AbstractThe heat shock protein 90 (Hsp90) is a molecular chaperone that employs the free energy of ATP hydrolysis to control the folding and activation of several client proteins in the eukaryotic cell. To elucidate how the local ATPase reaction in the active site couples to the global conformational dynamics of Hsp90, we integrate here large-scale molecular simulations with biophysical experiments. We show that the conformational switching of conserved ion pairs between the N-terminal domain, harbouring the active site, and the middle domain strongly modulates the catalytic barrier of the ATP-hydrolysis reaction by electrostatic forces. Our combined findings provide a mechanistic model for the coupling between catalysis and protein dynamics in Hsp90, and show how long-range coupling effects can modulate enzymatic activity.

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.

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