Three-Step Resonance Energy Transfer in Insulin Amyloid Fibrils

Tarabara, Uliana; Shchuka, Mykhailo; Vus, Kateryna; Zhytniakivska, Olga; Trusova, Valeriya; Gorbenko, Galyna; Gadjev, Nikolai; Deligeorgiev, Todor

doi:10.26565/2312-4334-2019-4-06

Cited by 3 publications

(19 citation statements)

References 41 publications

(56 reference statements)

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“…Materials Bovine insulin, dimethyl sulfoxide (DMSO), Tris, Thioflavin T (ThT) were purchased from Sigma. Benzanthrone dye ABM [21], and squaraine dyes, SQ1, SQ4 [19], were synthesized in the Daugavpils University and University of Sofia, respectively. All other reagents were used without further purification.…”

Section: Methodsmentioning

confidence: 99%

“…Insulin fibril samples for transmission microscopy were contrasted by 1.5% (w/v) phosphotungstic acid, dried and viewed with the EM-125 electron microscope (Selmi, Ukraine), as described previously [19].…”

Section: Preparation Of Working Solutionsmentioning

confidence: 99%

“…The efficiency of energy transfer was determined from the quenching of the donor fluorescence in the presence of acceptor using the theory described in our previous paper [19]. Briefly, the following formulas were used [1]:…”

Section: Spectroscopic Measurementsmentioning

confidence: 99%

“…where D I and DA I , are fluorescence intensities of the free donor and in presence of the acceptor, respectively. The donor fluorescence recorded upon addition of the acceptor was corrected for inner filter effect [19]. The Förster radius was calculated using the Mathcad 15.0 software and the equations:…”

Section: Spectroscopic Measurementsmentioning

confidence: 99%

“…Recently we have found that amyloid fibrils can also serve as a molecular scaffold for controlled positioning of a multitude of chromophores communicating via the two-step FRET [18,19]. Moreover, we have demonstrated that the technique based on the msFRET can be effectively used for the identification and characterization of insulin amyloid fibrils along with classical Thioflavin T (ThT) assay, thus allowing the increase of amyloid detection sensitivity [19,20].…”

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confidence: 99%

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Cascade Energy Transfer in Insulin Amyloid Fibrils Doped by Thioflavin T, Benzanthrone and Squarine Dyes

Tarabara

Vus

Shchuka

et al. 2020

EEJP

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The three-step Förster resonance energy transfer (FRET) within the cascade of four dyes, including the classical amyloid marker Thioflavin T as a primary donor, two jumper dyes, benzanthrone ABM and squaraine SQ4, and terminal acceptor SQ1, was tested as a possible tool for detection and characterization of insulin amyloid fibrils. The results obtained confirm the occurrence of highly efficient multistep FRET (msFRET) in the chromophore ensemble in the presence of insulin fibrils formed at elevated temperature under pH 2 (InsF1) or pH 7.4, 0.15 M NaCl (InsF2), while negligible FRET efficiencies were obtained for the control unfibrillized protein, suggesting the specificity of msFRET to cross-β-sheet architecture characteristic of amyloid fibrils. Specifically, the efficiencies of FRET for the donor-acceptor pairs ThT-ABM, ABM-SQ4 and SQ4-SQ1 at maximum acceptor concentrations (~0.4 µM – 1.6 µM) were estimated to be 86%/94%, 48%/34% and 66%/32%, respectively, in the presence of InsF1/InsF2. The most significant differences between InsF1/InsF2 and the control protein were observed for the donor-acceptor pair ThT-ABM, suggesting that ABM is the key mediator in the whole process of msFRET. Assuming the isotropic rotation of the fluorophores, the average donor-acceptor distances were estimated in the presence of InsF1, yielding the values 1.3 nm, 5.3 nm, and 3.9 nm for the ThT-ABM, ABM-SQ4 and SQ4-SQ1 pairs, respectively. The obtained distances are indicative of different fibril binding sites for the chromophores in the insulin fibrils, although due to their high specificity to the fibrillar structure, the dyes are most likely to localize in the surface grooves of β-sheets running along the main axis of amyloid fibril. Remarkably, the differences in the insulin amyloid morphology can be clearly distinguished using msFRET. As evidenced from TEM, InsF2 were thinner, shorter and contained amorphous aggregates, as compared to InsF1. Thus, different amyloid formation pathways under neutral and acidic pH resulted in the changes in the dye affinity for to the fibril binding sites, and, as a consequence, in the distinct msFRET efficiencies, especially for the pair SQ4-SQ1. The ability of ThT to serve as an efficient amplifier for the two near-infrared dyes, SQ4 and SQ1, with the benzanthrone fluorophore ABM as a jumper dye, allows detection of fibrillar insulin in the optical window of the biological samples, with the Stokes shift of the four-chromophore being ca. 240 nm. The proposed msFRET-based approach can be employed not only for insulin amyloid detection but also for distinguishing between different amyloid fibril morphologies and gaining further insights into the mechanisms involved in the development of the injection-localized insulin amyloidosis.

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Section: Methodsmentioning

confidence: 99%

Section: Preparation Of Working Solutionsmentioning

confidence: 99%

Section: Spectroscopic Measurementsmentioning

confidence: 99%

Section: Spectroscopic Measurementsmentioning

confidence: 99%

mentioning

confidence: 99%

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Cascade Energy Transfer in Insulin Amyloid Fibrils Doped by Thioflavin T, Benzanthrone and Squarine Dyes

Tarabara

Vus

Shchuka

et al. 2020

EEJP

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Binding of Benzanthrone Dye ABM to Insulin Amyloid Fibrils: Molecular Docking and Molecular Dynamics Simulation Studies

Vus

2020

EEJP

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The binding of the benzanthrone dye ABM to the model amyloid fibrils of human insulin, referred to here as vealyl (12-VEALYL-17, insulin B-chain)), lyqlen (13-LYQLEN-18, insulin A-chain) and Insf ( 11-LVEALYL-17, B-chain) + 12-SLYQLENY-19, A-chain) was studied by the molecular docking and molecular dynamics simulations. To obtain the relaxed structures with the enhanced conformational stability, the model fibril structures were solvated and equilibrated in water at 300-310 K using the Gromacs simulation package, with backbone position restraints being applied to prevent the beta-sheet disruption. It appeared that the vealyl fibril relaxation resulted in the twisting of the two β-sheets, and only the vealyl fibril remained stable during 20 ns MD simulations of the relaxed structures. Next, Insf, vealyl, lyqlen, and vealyl (relaxed) fibrils were used for the molecular docking studies (by SwissDock), revealing the binding modes of ABM and standard amyloid marker Thioflavin T (ThT) to the examined fibril structures. Specifically, in the most energetically stable complex the vealyl (relaxed) fibril binding site for ABM was located on the dry steric zipper interface, although the dye was associated with only one twisted β-sheet. During the 20 ns MD simulation the ABM fibril location was changed to a deeper position in the dry interface between the two β-sheets, with the dye-interacting residues being represented by 6 LEU, 3 VAL, 2 ALA, 1 TYR and 1 GLU. The binging free energy Δ(Gbinding) for ABM complexation with vealyl (relaxed) fibril evaluated with the GMXPBSA GROMACS tool was found to be –31.4±1.8 kJ/mol, that is in accordance with our estimates derived from the fluorescence studies for ABM binding to the bovine insulin amyloid fibrils Δ(Gbinding)= –30.2 kJ/mol. The Lennard-Jones component appeared to dominate the dye-fibril interactions, with much smaller contributions of Coulombic and nonpolar solvation terms to the total Δ(Gbinding) value, and unfavorable effect of the polar solvation term. These findings indicate that a high specificity of ABM to the insulin amyloid fibrils may arise predominantly from the dye-protein hydrophobic interactions, followed by the formation of van der Waals contacts, thus providing additional evidence for sensitivity of the dye spectral properties to environmental polarity, suggested in our previous studies. Overall, the obtained results provided further insights into the atomistic mechanism of the ABM binding to insulin amyloid fibrils and can be used for development of the novel fluorescent reporters possessing high sensitivity to the amyloid assemblies.

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Multiple Docking of Fluorescent Dyes to Fibrillar Insulin

Tarabara

Zhytniakivska

Trusova

et al. 2022

EEJP

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The co-localization of the donor and acceptor fluorophores capable of transferring the energy by the Fӧrster mechanism, on the molecular scaffold of amyloid fibrils offers new opportunities not only for refinement of the amyloid detection and structural analysis, but also for designing photonic nanodevices on their basis. The assembly of these systems involves the non-covalent dye-protein interactions which can hardly be characterized in terms of a precise dye location within the fibril structure that is required for fabricating the FRET-based light harvesting systems or photonic nanowires. In view of this, the dye-fibril binding process deserves a detailed in silico study. In the previous molecular docking studies of the FRET donors and acceptors interacting with the insulin model fibrils we considered only one ligand during the simulation procedure. However, the real situation is much more complicated, when the multiple ligands can compete for the same binding site, a direct complexation between the dyes on the fibril scaffold can take place, the spatial distribution of the bound fluorophores can be unfavorable for the energy transfer, etc. In addition, the mutual orientation of the donor and acceptor molecules essentially contribute to the efficiency of the Förster resonance energy transfer (FRET) in the investigated systems. The present study was undertaken to gain molecular docking insight into the binding of the donor (Thioflavin T) and acceptor (Congo Red or a phosphonium dye TDV) fluorophores to the insulin amyloid fibrils using the multiple docking approach. The employed PatcDock and SwissDock webserves provided evidence for the preferable association of all dyes with the fibril grooves. The protein-ligand interaction profiler (PLIP) was employed for analyzing the (InsF + ThT +CR) and (InsF + ThT + TDV) systems. The revealed binding modes and the types of the dye-fibril interactions may be of importance for a more detailed analysis of the FRET process in amyloid systems and may serve as a background for further in silico studies of the cascade FRET on the amyloid fibril scaffold.

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Three-Step Resonance Energy Transfer in Insulin Amyloid Fibrils

Cited by 3 publications

References 41 publications

Cascade Energy Transfer in Insulin Amyloid Fibrils Doped by Thioflavin T, Benzanthrone and Squarine Dyes

Cascade Energy Transfer in Insulin Amyloid Fibrils Doped by Thioflavin T, Benzanthrone and Squarine Dyes

Binding of Benzanthrone Dye ABM to Insulin Amyloid Fibrils: Molecular Docking and Molecular Dynamics Simulation Studies

Multiple Docking of Fluorescent Dyes to Fibrillar Insulin

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