Browse
Submit Data
Databases
API
Help

Dataset Information

19 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Single-molecule FRET uncovers hidden conformations and dynamics of human Argonaute 2.

ABSTRACT: Human Argonaute 2 (hAgo2) constitutes the functional core of the RNA interference pathway. Guide RNAs direct hAgo2 to target mRNAs, which ultimately leads to hAgo2-mediated mRNA degradation or translational inhibition. Here, we combine site-specifically labeled hAgo2 with time-resolved single-molecule FRET measurements to monitor conformational states and dynamics of hAgo2 and hAgo2-RNA complexes in solution that remained elusive so far. We observe dynamic anchoring and release of the guide's 3'-end from the PAZ domain during the stepwise target loading process even with a fully complementary target. We find differences in structure and dynamic behavior between partially and fully paired canonical hAgo2-guide/target complexes and the miRNA processing complex formed by hAgo2 and pre-miRNA451. Furthermore, we detect a hitherto unknown conformation of hAgo2-guide/target complexes that poises them for target-directed miRNA degradation. Taken together, our results show how the conformational flexibility of hAgo2-RNA complexes determines function and the fate of the ribonucleoprotein particle.

SUBMITTER: Willkomm S

PROVIDER: S-EPMC9250533 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Manipulating protein conformations by single-molecule AFM-FRET nanoscopy.

Project description:Combining atomic force microscopy and fluorescence resonance energy transfer spectroscopy (AFM-FRET), we have developed a single-molecule AFM-FRET nanoscopy approach capable of effectively pinpointing and mechanically manipulating a targeted dye-labeled single protein in a large sampling area and simultaneously monitoring the conformational changes of the targeted protein by recording single-molecule FRET time trajectories. We have further demonstrated an application of using this nanoscopy on manipulation of single-molecule protein conformation and simultaneous single-molecule FRET measurement of a Cy3-Cy5-labeled kinase enzyme, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase). By analyzing time-resolved FRET trajectories and correlated AFM force pulling curves of the targeted single-molecule enzyme, we are able to observe the protein conformational changes of a specific coordination by AFM mechanic force pulling.

| S-EPMC3662055 | biostudies-literature

Single-molecule FRET supports the two-state model of Argonaute action.

Project description:Argonaute can be found in all three domains of life and is the functional core of the eukaryotic RNA-silencing machinery. In order to shed light on the conformational changes that drive Argonaute action, we performed single-molecule FRET measurements employing a so far uncharacterized member of the Argonaute family, namely Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo). We show that MjAgo is a catalytically active Argonaute variant hydrolyzing exclusively DNA target strands out of a DNA/DNA hybrid. We studied the interplay between Argonaute and nucleic acids using fluorescent dyes covalently attached at different positions of the DNA guide as steric reporters. This allowed us to determine structurally confined parts of the protein scaffold and flexible regions of the DNA guide. Single-molecule FRET measurements demonstrate that the 3'end of the DNA guide is released from the PAZ domain upon target strand loading. This conformational change does not necessitate target strand cleavage but a fully complementary target strand. Thus, our data support the two state model for Argonaute action.

| S-EPMC3929424 | biostudies-literature

Single-molecule FRET reveals hidden complexity in a protein energy landscape.

Project description:Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured "safety pin" or "staple" from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.

| S-EPMC4291146 | biostudies-literature

Complex RNA folding kinetics revealed by single-molecule FRET and hidden Markov models.

Project description:We have developed a hidden Markov model and optimization procedure for photon-based single-molecule FRET data, which takes into account the trace-dependent background intensities. This analysis technique reveals an unprecedented amount of detail in the folding kinetics of the Diels-Alderase ribozyme. We find a multitude of extended (low-FRET) and compact (high-FRET) states. Five states were consistently and independently identified in two FRET constructs and at three Mg(2+) concentrations. Structures generally tend to become more compact upon addition of Mg(2+). Some compact structures are observed to significantly depend on Mg(2+) concentration, suggesting a tertiary fold stabilized by Mg(2+) ions. One compact structure was observed to be Mg(2+)-independent, consistent with stabilization by tertiary Watson-Crick base pairing found in the folded Diels-Alderase structure. A hierarchy of time scales was discovered, including dynamics of 10 ms or faster, likely due to tertiary structure fluctuations, and slow dynamics on the seconds time scale, presumably associated with significant changes in secondary structure. The folding pathways proceed through a series of intermediate secondary structures. There exist both compact pathways and more complex ones, which display tertiary unfolding, then secondary refolding, and, subsequently, again tertiary refolding.

| S-EPMC3977575 | biostudies-literature

Single-molecule FRET reveals sugar-induced conformational dynamics in LacY.

Project description:The N- and C-terminal six-helix bundles of lactose permease (LacY) form a large internal cavity open on the cytoplasmic side and closed on the periplasmic side with a single sugar-binding site at the apex of the cavity near the middle of the molecule. During sugar/H(+) symport, an outward-facing cavity is thought to open with closing of the inward-facing cavity so that the sugar-binding site is alternately accessible to either face of the membrane. In this communication, single-molecule fluorescence (Förster) resonance energy transfer is used to test this model with wild-type LacY and a conformationally restricted mutant. Pairs of Cys residues at the ends of two helices on the cytoplasmic or periplasmic sides of wild-type LacY and the mutant were labeled with appropriate donor and acceptor fluorophores, single-molecule fluorescence resonance energy transfer was determined in the absence and presence of sugar, and distance changes were calculated. With wild-type LacY, binding of a galactopyranoside, but not a glucopyranoside, results in a decrease in distance on the cytoplasmic side and an increase in distance on the periplasmic side. In contrast, with the mutant, a more pronounced decrease in distance and in distance distribution is observed on the cytoplasmic side, but there is no change on the periplasmic side. The results are consistent with the alternating access model and indicate that the defect in the mutant is due to impaired ligand-induced flexibility on the periplasmic side.

| S-EPMC1937519 | biostudies-other

Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1?.

Project description:The dynamic architecture of chromatin fibers, a key determinant of genome regulation, is poorly understood. Here, we employ multimodal single-molecule Förster resonance energy transfer studies to reveal structural states and their interconversion kinetics in chromatin fibers. We show that nucleosomes engage in short-lived (micro- to milliseconds) stacking interactions with one of their neighbors. This results in discrete tetranucleosome units with distinct interaction registers that interconvert within hundreds of milliseconds. Additionally, we find that dynamic chromatin architecture is modulated by the multivalent architectural protein heterochromatin protein 1? (HP1?), which engages methylated histone tails and thereby transiently stabilizes stacked nucleosomes. This compacted state nevertheless remains dynamic, exhibiting fluctuations on the timescale of HP1? residence times. Overall, this study reveals that exposure of internal DNA sites and nucleosome surfaces in chromatin fibers is governed by an intrinsic dynamic hierarchy from micro- to milliseconds, allowing the gene regulation machinery to access compact chromatin.

| S-EPMC5770380 | biostudies-literature

Potassium channel selectivity filter dynamics revealed by single-molecule FRET.

Project description:Potassium (K) channels exhibit exquisite selectivity for conduction of K+ ions over other cations, particularly Na+. High-resolution structures reveal an archetypal selectivity filter (SF) conformation in which dehydrated K+ ions, but not Na+ ions, are perfectly coordinated. Using single-molecule FRET (smFRET), we show that the SF-forming loop (SF-loop) in KirBac1.1 transitions between constrained and dilated conformations as a function of ion concentration. The constrained conformation, essential for selective K+ permeability, is stabilized by K+ but not Na+ ions. Mutations that render channels nonselective result in dilated and dynamically unstable conformations, independent of the permeant ion. Further, while wild-type KirBac1.1 channels are K+ selective in physiological conditions, Na+ permeates in the absence of K+. Moreover, whereas K+ gradients preferentially support 86Rb+ fluxes, Na+ gradients preferentially support 22Na+ fluxes. This suggests differential ion selectivity in constrained versus dilated states, potentially providing a structural basis for this anomalous mole fraction effect.

| S-EPMC6430689 | biostudies-literature

Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA.

Project description:Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3' terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3' terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3' cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3' termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3' terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3' vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.

| S-EPMC5137447 | biostudies-literature

Variational Bayes analysis of a photon-based hidden Markov model for single-molecule FRET trajectories.

Project description:Single-molecule fluorescence resonance energy transfer (smFRET) measurement is a powerful technique for investigating dynamics of biomolecules, for which various efforts have been made to overcome significant stochastic noise. Time stamp (TS) measurement has been employed experimentally to enrich information within the signals, while data analyses such as the hidden Markov model (HMM) have been successfully applied to recover the trajectories of molecular state transitions from time-binned photon counting signals or images. In this article, we introduce the HMM for TS-FRET signals, employing the variational Bayes (VB) inference to solve the model, and demonstrate the application of VB-HMM-TS-FRET to simulated TS-FRET data. The same analysis using VB-HMM is conducted for other models and the previously reported change point detection scheme. The performance is compared to other analysis methods or data types and we show that our VB-HMM-TS-FRET analysis can achieve the best performance and results in the highest time resolution. Finally, an smFRET experiment was conducted to observe spontaneous branch migration of Holliday-junction DNA. VB-HMM-TS-FRET was successfully applied to reconstruct the state transition trajectory with the number of states consistent with the nucleotide sequence. The results suggest that a single migration process frequently involves rearrangement of multiple basepairs.

| S-EPMC3446682 | biostudies-literature

Structural dynamics of potassium-channel gating revealed by single-molecule FRET.

Project description:Crystallography has provided invaluable insights regarding ion-channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites and then examined structural dynamics within lipid membranes by single-molecule fluorescence resonance energy transfer (FRET). We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states, both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and in cation channels in general.

| S-EPMC4833211 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data