scholarly journals Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA and actin filaments

2017 ◽  
Author(s):  
Sofia Espinoza-Sanchez ◽  
Lauren Ann Metskas ◽  
Steven Z. Chou ◽  
Elizabeth Rhoades ◽  
Thomas D. Pollard
Keyword(s):  
Electron Microscopy ◽  
Energy Transfer ◽  
Structural Change ◽  
Fluorescence Spectroscopy ◽  
Conformational Changes ◽  
Actin Filaments ◽  
Fluorescent Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fret Efficiency

AbstractWe used fluorescence spectroscopy and electron microscopy to determine how binding of ATP, nucleation-promoting factors (NPF), actin monomers and actin filaments change the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C-termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ETeff) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide binding clefts of the Arp subunits. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ETeff between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected a different structural change that involves bringing ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. Each of the ligands that activates Arp2/3 complex changes the structure in different ways, each leading progressively to fully activated Arp2/3 complex on the side of a filament.

scholarly journals Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments

2018 ◽  
Vol 115 (37) ◽  
pp. E8642-E8651 ◽  
Author(s):  
Sofia Espinoza-Sanchez ◽  
Lauren Ann Metskas ◽  
Steven Z. Chou ◽  
Elizabeth Rhoades ◽  
Thomas D. Pollard
Keyword(s):  
Energy Transfer ◽  
Structural Change ◽  
Actin Filament ◽  
Conformational Changes ◽  
Actin Filaments ◽  
Fluorescent Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fret Efficiency ◽  
Binding Cleft

We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ETeff) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ETeff between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament.

scholarly journals Biomimetic Synthesis, Characterization, and Evaluation of Fluorescence Resonance Energy Transfer, Photoluminescence, and Photocatalytic Activity of Zinc Oxide Nanoparticles

2021 ◽  
Vol 13 (4) ◽  
pp. 2004
Author(s):  
Udari Wijesinghe ◽  
Gobika Thiripuranathar ◽  
Haroon Iqbal ◽  
Farid Menaa
Keyword(s):  
Electron Microscopy ◽  
Zinc Oxide ◽  
Photocatalytic Activity ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Solar Irradiation ◽  
Zno Nps ◽  
Fluorescence Resonance

Owing to the development of nanotechnology, biosynthesis of nanoparticles (NPs) is gaining considerable attention as a cost-effective and eco-friendly approach that minimizes the effects of toxic chemicals used in NP fabrication. The present work reports low-cost phytofabrication of zinc oxide (ZnO) NPs employing aqueous extracts of various parts (leaves, stems, and inflorescences) of Tephrosia purpurea (T. purpurea). The formation, structure, morphology, and other physicochemical properties of ZnO NPs were characterized by ultraviolet–visible (UV–Vis) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, and dynamic light scattering (DLS). UV–Vis spectral analysis revealed sharp surface plasmon resonance (SPR) at around 250–280 nm, while the XRD patterns confirmed distinctive peaks indices to the crystalline planes of hexagonal wurtzite ZnO NPs. TEM analysis confirmed the presence of spherical-shaped ZnO NPs with average particle sizes (PS) between 25–35 nm, which was in agreement with the XRD results. FTIR analysis revealed that phenolics, flavonoids, amides, alkaloids, and amines present in the plant extract are responsible for the stabilization of the ZnO NPs. Further, the hydrodynamic diameter in the range of 85–150 nm was measured using the DLS technique. The fluorescence resonance energy transfer (FRET) ability of biogenic ZnO NPs was evaluated, and the highest efficiency was found in ZnO NPs synthesized via T. purpurea inflorescences extract. Photoluminescence (PL) spectra of biogenic ZnO NPs showed three emission peaks consisting of a UV–Vis region with high-intensity compared to that of chemically synthesized ZnO NPs. The biosynthesized ZnO NPs showed photocatalytic activity under solar irradiation by enhancing the degradation rate of methylene blue (MB). Among the prepared biogenic ZnO NPs, T. purpurea leaves mediated with NPs acted as the most effective photocatalyst, with a maximum degradation efficiency of 98.86% and a half-life of 84.7 min. This is the first report related to the synthesis of multifunctional ZnO NPs using T. purpurea, with interesting characteristics for various potential applications in the future.

Structural rearrangements of the motor protein prestin revealed by fluorescence resonance energy transfer

2009 ◽  
Vol 297 (2) ◽  
pp. C290-C298 ◽  
Author(s):  
Kristin Rule Gleitsman ◽  
Michihiro Tateyama ◽  
Yoshihiro Kubo
Keyword(s):  
Energy Transfer ◽  
Membrane Potential ◽  
Fluorescence Resonance Energy Transfer ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Motor Protein ◽  
Resonance Energy ◽  
Outer Hair Cells ◽  
Nonlinear Capacitance ◽  
Fluorescence Resonance

Prestin is a membrane protein expressed in the outer hair cells (OHCs) in the cochlea that is essential for hearing. This unique motor protein transduces a change in membrane potential into a considerable mechanical force, which leads to a cell length change in the OHC. The nonlinear capacitance in cells expressing prestin is recognized to reflect the voltage-dependent conformational change of prestin, of which its precise nature remains unknown. In the present work, we aimed to detect the conformational changes of prestin by a fluorescence resonance energy transfer (FRET)-based technique. We heterologously expressed prestin labeled with fluorophores at the COOH- or NH2-terminus in human embryonic kidney-293T cells, and monitored FRET changes on depolarization-inducing high KCl application. We detected a significant decrease in intersubunit FRET both between the COOH-termini and between the COOH- and NH2-termini. A similar FRET decrease was observed when membrane potential was directly and precisely controlled by simultaneous patch clamp. Changes in FRET were suppressed by either of two treatments known to abolish nonlinear capacitance, V499G/Y501H mutation and sodium salicylate. Our results are consistent with significant movements in the COOH-terminal domain of prestin upon change in membrane potential, providing the first dynamic information on its molecular rearrangements.

Fluorescent resonance energy transfer -based biosensor for detecting conformational changes of Pin1

2018 ◽  
Vol 505 (2) ◽  
pp. 399-404 ◽  
Author(s):  
Masafumi Hidaka ◽  
Emiko Okabe ◽  
Kodai Hatakeyama ◽  
Heather Zook ◽  
Chiyoko Uchida ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescent Resonance Energy Transfer

Fluorescence resonance energy transfer in ferritin labeled with multiple fluorescent dyes

2007 ◽  
Vol 13 (3) ◽  
pp. 349-355 ◽  
Author(s):  
Belén Fernández ◽  
Natividad Gálvez ◽  
Purificación Sánchez ◽  
Rafael Cuesta ◽  
Ruperto Bermejo ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Resonance
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