Search results for "SINGLE-MOLECULE FRET"
showing 8 items of 8 documents
Probing Differential Binding Mechanisms of Phenylalanine-Glycine-Rich Nucleoporins by Single-Molecule FRET
2018
Abstract Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex. They are highly dynamic under physiological conditions and studying their interaction with nuclear transport receptors (NTRs) is key to understanding the molecular mechanism of nucleocytoplasmic transport. Distinct conformational features of FG-Nups interacting with diverse NTRs can be detected by multiparameter single-molecule fluorescence energy transfer (smFRET), which is a powerful technique for studying the dynamics and interactions of biomolecules in solution. Here we provide a detailed protocol utilizing smFRET to reveal dif…
Two differential binding mechanisms of FG-nucleoporins and nuclear transport receptors
2018
Summary Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex (NPC). Previous studies showed that nuclear transport receptors (NTRs) were found to interact with FG-Nups by forming an “archetypal-fuzzy” complex through the rapid formation and breakage of interactions with many individual FG motifs. Here, we use single-molecule studies combined with atomistic simulations to show that, in sharp contrast, FG-Nup214 undergoes a coupled reconfiguration-binding mechanism when interacting with the export receptor CRM1. Association and dissociation rate constants are more than an order of magnitude lowe…
Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study
2018
Single-molecule Forster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between +/- 0.02 and +/- 0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and…
FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.
2021
International audience; Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever- increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among d…
Single-Molecule FRET Reveals a Cooperative Effect of Two Methyl Group Modifications in the Folding of Human Mitochondrial tRNALys
2011
Summary Using a combination of advanced RNA synthesis techniques and single molecule spectroscopy, the deconvolution of individual contributions of posttranscriptional modifications to the overall folding and stabilization of human mitochondrial tRNA Lys is described. An unexpected destabilizing effect of two pseudouridines on the native tRNA folding was evidenced. Furthermore, the presence of m 2 G10 alone does not facilitate the folding of tRNA Lys , but a stabilization of the biologically functional cloverleaf shape in conjunction with the principal stabilizing component m 1 A9 exceeds the contribution of m 1 A alone. This constitutes an unprecedented cooperative effect of two nucleotide…
Inside a Shell—Organometallic Catalysis Inside Encapsulin Nanoreactors
2021
Abstract Compartmentalization of chemical reactions inside cells are a fundamental requirement for life. Encapsulins are self‐assembling protein‐based nanocompartments from the prokaryotic repertoire that present a highly attractive platform for intracellular compartmentalization of chemical reactions by design. Using single‐molecule Förster resonance energy transfer and 3D‐MINFLUX analysis, we analyze fluorescently labeled encapsulins on a single‐molecule basis. Furthermore, by equipping these capsules with a synthetic ruthenium catalyst via covalent attachment to a non‐native host protein, we are able to perform in vitro catalysis and go on to show that engineered encapsulins can be used …
Single-molecule FRET studies of counterion effects on the free energy landscape of human mitochondrial lysine tRNA.
2011
The folding energy landscape of RNA is greatly affected by interactions between the RNA and counterions that neutralize the backbone negative charges and may also participate in tertiary contacts. Valence, size, coordination number, and electron shell structure can all contribute to the energetic stabilization of specific RNA conformations. Using single-molecule fluorescence resonance energy transfer (smFRET), we have examined the folding properties of the RNA transcript of human mitochondrial tRNA(Lys), which possesses two different folded states in addition to the unfolded one under conditions of thermodynamic equilibrium. We have quantitatively analyzed the degree of RNA tertiary structu…
Conformational Dynamics of the Dengue Virus Protease Revealed by Fluorescence Correlation and Single-Molecule FRET Studies.
2021
The dengue virus protease (DENV-PR) represents an attractive target for counteracting DENV infections. It is generally assumed that DENV-PR can exist in an open and a closed conformation and that active site directed ligands stabilize the closed state. While crystal structures of both the open and the closed conformation were successfully resolved, information about the prevalence of these conformations in solution remains elusive. Herein, we address the question of whether there is an equilibrium between different conformations in solution which can be influenced by addition of a competitive inhibitor. To this end, DENV-PR was statistically labeled by two dye molecules constituting a FRET …