Search results for "Förster resonance energy transfer"
showing 10 items of 70 documents
Aza-macrocyclic triphenylamine ligands for G-quadruplex recognition
2018
A new series of triphenylamine-based ligands with one (TPA1PY), two (TPA2PY) or three pendant aza-macrocycle(s) (TPA3PY) has been synthesised and studied by means of pH-metric titrations, UV/Vis spectroscopy and fluorescence experiments. The affinity of these ligands for G-quadruplex (G4) DNA and the selectivity they show for G4s over duplex DNA were investigated by Forster resonance energy transfer (FRET) melting assays, fluorimetric titrations and circular dichroism spectroscopy. Interestingly, the interactions of the bi- and especially the tri-branched ligands with G4s lead to a very intense redshifted fluorescence emission band that may be associated with intermolecular aggregation betw…
FRET biosensor allows spatio-temporal observation of shear stress-induced polar RhoGDIα activation
2018
Rho GDP-dissociation inhibitor α (RhoGDIα) is a known negative regulator of the Rho family that shuts off GDP/GTP cycling and cytoplasm/membrane translocation to regulate cell migration. However, to our knowledge, no reports are available that focus on how the RhoGDIα-Rho GTPases complex is activated by laminar flow through exploring the activation of RhoGDIα itself. Here, we constructed a new biosensor using fluorescence resonance energy transfer (FRET) technology to measure the spatio-temporal activation of RhoGDIα in its binding with Rho GTPases in living HeLa cells. Using this biosensor, we find that the dissociation of the RhoGDIα-Rho GTPases complex is increased by shear stress, and i…
Conformational dynamics of a single protein monitored for 24 hours at video rate
2018
We use plasmon rulers to follow the conformational dynamics of a single protein for up to 24 h at a video rate. The plasmon ruler consists of two gold nanospheres connected by a single protein linker. In our experiment, we follow the dynamics of the molecular chaperone heat shock protein 90 (Hsp90), which is known to show “open” and “closed” conformations. Our measurements confirm the previously known conformational dynamics with transition times in the second to minute time scale and reveals new dynamics on the time scale of minutes to hours. Plasmon rulers thus extend the observation bandwidth 3–4 orders of magnitude with respect to single-molecule fluorescence resonance energy transfer a…
Polysialic acid chains exhibit enhanced affinity for ordered regions of membranes.
2018
Polysialic acid (polySia) forms linear chains which are usually attached to the external surface of the plasma membrane mainly through the Neural Cell Adhesion Molecule (NCAM) protein. It is exposed on neural cells, several types of cancer cells, dendritic cells, and egg and sperm cells. There are several lipid raft-related phenomena in which polySia is involved; however the mechanisms of polySia action as well as determinants of its localization in lipid raft microdomains are still unknown, although the majority of NCAM molecules in the liquid-ordered raft membrane fractions of neural cells appear to be polysialylated. Here we investigate the affinity of polySia (both soluble and NCAM-depe…
FRET-based method for evaluation of the efficiency of reversible and irreversible sonoporation.
2017
It is widely known that not all of the treated cells survive after introduction of exogenous molecules via any physical method. Therefore, it is important to develop methods that would allow simultaneous evaluation of both molecular delivery efficiency and cell viability. This study presents Forster resonance energy transfer (FRET)-based method that allows molecular transfer and cell viability evaluation in a single measurement by employing two common fluorescent dyes, namely, ethidium bromide and trypan blue. The method has been validated using cell sonoporation. The FRET-based method allows the efficiency evaluation of both reversible and irreversible sonoporation in a single experiment. …
Kinetic evidence for interaction of TMPyP4 with two different G-quadruplex conformations of human telomeric DNA
2018
Background: Stabilization of G-quadruplex helices by small ligands has attracted growing attention because they inhibit the activity of the enzyme telomerase, which is overexpressed in> 80% cancer cells. TMPyP4, one of the most studied G-quadruplex ligands, is used as a model to show that the ligands can exhibit different binding features with different conformations of a human telomeric specific sequence. Methods: UV–Vis, FRET melting Assay, Isothermal Titration Calorimetry, Time-resolved Fluorescence lifetime, T-Jump and Molecular Dynamics. Results: TMPyP4 yields two different complexes with two Tel22 telomeric conformations in the presence of Na+ or K+. T-Jump kinetic experiments show th…
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…
Comment on “Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water”
2018
Editors at Science requested our input on the above discussion (comment by Best et al . and response by Riback et al .) because both sets of authors use our data from Fuertes et al . (2017) to support their arguments. The topic of discussion pertains to the discrepant inferences drawn from SAXS versus FRET measurements regarding the dimensions of intrinsically disordered proteins (IDPs) in aqueous solvents. Using SAXS measurements on labeled and unlabeled proteins, we ruled out the labels used for FRET measurements as the cause of discrepant inferences between the two methods. Instead, we propose that FRET and SAXS provide complementary readouts because of a decoupling of size and shape fl…
Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers
2018
Influenza hemagglutinin (HA) is the canonical type I viral envelope glycoprotein and provides a template for the membrane-fusion mechanisms of numerous viruses. The current model of HA-mediated membrane fusion describes a static "spring-loaded" fusion domain (HA2) at neutral pH. Acidic pH triggers a singular irreversible conformational rearrangement in HA2 that fuses viral and cellular membranes. Here, using single-molecule Förster resonance energy transfer (smFRET)-imaging, we directly visualized pH-triggered conformational changes of HA trimers on the viral surface. Our analyses reveal reversible exchange between the pre-fusion and two intermediate conformations of HA2. Acidification of p…
Peripherin-2 differentially interacts with cone opsins in outer segments of cone photoreceptors
2016
Peripherin-2 is a glycomembrane protein exclusively expressed in the light-sensing compartments of rod and cone photoreceptors designated as outer segments (OS). Mutations in peripherin-2 are associated with degenerative retinal diseases either affecting rod or cone photoreceptors. While peripherin-2 has been extensively studied in rods, there is only little information on its supramolecular organization and function in cones. Recently, we have demonstrated that peripherin-2 interacts with the light detector rhodopsin in OS of rods. It remains unclear, however, if peripherin-2 also binds to cone opsins. Here, using a combination of co-immunoprecipitation analyses, transmission electron micr…