Rotational and Translational Diffusion of Low Molecular Weight Nanoprobes in Ficoll Solutions

Jhamba, Elton; M'Rah, Zakaria; Boukari, Hacène

doi:10.2174/1573413713666170110110938

Cited by 3 publications

(2 citation statements)

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“…It is worth mentioning that Ficoll may start to entangle, forming a meshlike environment at high concentration (∼500 g/L) as determined using dynamic light scattering. 66 However, the concentration range used in the work presented here (0−300 g/L) is below the threshold for mesh formation.…”

Section: Resultsmentioning

confidence: 85%

“…In addition, the water-soluble Ficoll-70 (70 kDa, neutral, branched polysaccharide) was chosen specifically to minimize electrostatic interactions with the FRET probes, which allows us to understand how confinement and excluded volumes may influence energy-transfer efficiencies and the rotational dynamics via steric hindrance. It is worth mentioning that Ficoll may start to entangle, forming a meshlike environment at high concentration (∼500 g/L) as determined using dynamic light scattering . However, the concentration range used in the work presented here (0–300 g/L) is below the threshold for mesh formation.…”

Section: Results and Discussionmentioning

confidence: 98%

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Crowding Effects on Energy-Transfer Efficiencies of Hetero-FRET Probes As Measured Using Time-Resolved Fluorescence Anisotropy

et al. 2018

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Macromolecular crowding is prevalent in all living cells due to the presence of large biomolecules and organelles. Cellular crowding is heterogeneous and is known to influence biomolecular transport, biochemical reactions, and protein folding. Emerging evidence suggests that some cell pathologies may be correlated with compartmentalized crowding. As a result, there is a need for robust biosensors that are sensitive to crowding as well as quantitative, noninvasive fluorescence methods that are compatible with living cells studies. Here, we have developed a model that describes the rotational dynamics of hetero-Förster resonance energy transfer (FRET) biosensors as a means to determine the energy-transfer efficiency and donor–acceptor distance. The model was tested on wavelength-dependent time-resolved fluorescence anisotropy of hetero-FRET probes (mCerulean3–linker–mCitrine) with variable linkers in both crowded (Ficoll-70) and viscous (glycerol) solutions at room temperature. Our results indicate that the energy-transfer efficiencies of these FRET probes increase as the linker becomes shorter and more flexible in pure buffer at room temperature. In addition, the FRET probes favor compact structures with enhanced energy-transfer efficiencies and a shorter donor–acceptor distance in the heterogeneous, polymer-crowded environment due to steric hindrance. In contrast, the extended conformation of these FRET probes is more favorable in viscous, homogeneous environments with a reduced energy-transfer efficiency compared to those in pure buffer, which we attribute to reduced structural fluctuations of the mCerulean3–mCitrine FRET pair in the glycerol-enriched buffer. Our results represent an important step toward the application of quantitative and noninvasive time-resolved fluorescence anisotropy of hetero-FRET probes to investigate compartmentalized macromolecular crowding and protein–protein interactions in living cells as well as in controlled environments.

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Section: Resultsmentioning

confidence: 85%

Section: Results and Discussionmentioning

confidence: 98%

Crowding Effects on Energy-Transfer Efficiencies of Hetero-FRET Probes As Measured Using Time-Resolved Fluorescence Anisotropy

et al. 2018

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Rotational and translational diffusion of size-dependent fluorescent probes in homogeneous and heterogeneous environments

Lee

Cong

Leopold

et al. 2018

Phys. Chem. Chem. Phys.

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Living cells are crowded with dynamic distributions of macromolecules and organelles that influence protein diffusion, molecular transport, biochemical reactions, and protein assembly. Here, we test the hypothesis that the diffusion of single molecules deviates from Brownian motion as described by the Stokes-Einstein model in a manner that depends on the viscosity range, the chemical structure of both the diffusing species and the crowding agents, and the spatio-temporal resolution of the employed analytical methods. Our size-dependent fluorescent probes are rhodamine-110, quantum dots, enhanced green fluorescent proteins (EGFP), and mCerulean3-linker-mCitrine FRET probes with various linker length and flexibility. Using fluorescence correlation spectroscopy (FCS), we investigated the translational diffusion of structure-dependent fluorescent probes, at the single-molecule level, in homogeneous (glycerol) and heterogeneous (Ficoll-70) solutions as a function of the bulk viscosity. Complementary rotational diffusion studies using time-resolved anisotropy enable us to assess weak interactions in crowded and viscous environments. Overall, our results show negative deviation from the Stokes-Einstein model in a fluorophore- and environment-dependent manner. In addition, the deviation between the FCS-measured hydrodynamic radius of the FRET probes in a buffer at room temperature and the molecular-weight based estimate (Perrin equation) as the number of the amino acid residues in the linker increases. These studies are essential for quantitative biophysics using fluorescence- and diffusion-based studies of protein-protein interactions and biomolecular transport in living cells.

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Diffusion of fluorescent poly(vinyl-alcohol) linear chains in semi-dilute poly(vinyl-alcohol) polymeric solutions

Boukari¹,

Sackett²,

Nossal³

2018

Biophysics, Biology and Biophotonics III: The Crossroads

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Rotational and Translational Diffusion of Low Molecular Weight Nanoprobes in Ficoll Solutions

Cited by 3 publications

References 0 publications

Crowding Effects on Energy-Transfer Efficiencies of Hetero-FRET Probes As Measured Using Time-Resolved Fluorescence Anisotropy

Crowding Effects on Energy-Transfer Efficiencies of Hetero-FRET Probes As Measured Using Time-Resolved Fluorescence Anisotropy

Rotational and translational diffusion of size-dependent fluorescent probes in homogeneous and heterogeneous environments

Diffusion of fluorescent poly(vinyl-alcohol) linear chains in semi-dilute poly(vinyl-alcohol) polymeric solutions

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