Ultrafast fluorescence spectroscopy reveals a dominant weakly-emissive population of fibril bound thioflavin-T

Singh, Prabhat; Mora, Aruna K.; Nath, Sukhendu

doi:10.1039/c5cc04256a

Cited by 47 publications

(76 citation statements)

References 26 publications

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“…The binding constants thus estimated from the fitting, were found to be 1.0 10 6 and 4.1 10 4 m À1 .E arlier,T hT was reported to follow two modes of binding, one in the fibrillar channel and the other in the grooves of the fibrillars urface. [42][43][44] Due to structural complementarity between ThT and the fibrillar channel, the binding in the inner channel of the fibrils is very strong compared to that on the surface. Considering the similar chemical structure of ThT and 2Me-DABT,i na nalogy,w ea lso propose that the stronger binding mode corresponds to the 2Me-DABT bound to the inner channel of the fibril and the weaker mode is due to the binding of 2Me-DABT ontot he fibrillar surface.…”

Section: Resultsmentioning

confidence: 99%

Benzothiazole‐Based Neutral Ratiometric Fluorescence Sensor for Amyloid Fibrils

Mora

Murudkar

Alamelu

et al. 2016

Chemistry A European J

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Early detection of amyloid fibrils is very important for the timely diagnosis of several neurological diseases. Thioflavin-T (ThT) is a gold standard fluorescent probe for amyloid fibrils and has been used for the last few decades. However, due to its positive charge, ThT is incapable of crossing the blood-brain barrier and cannot be used for in vivo imaging of fibrils. In the present work, we synthesized a neutral ThT derivative, 2-[2'-Me,4'-(dimethylamino)phenyl]benzothiazole (2Me-DABT), which showed a strong affinity towards the amyloid fibrils. On association with the amyloid fibrils, 2Me-DABT not only showed a large increase in its emission intensity, but also, unlike ThT, a large blueshift in its emission spectrum was observed. Thus, unlike ThT, 2Me-DABT is a potential candidate for the ratiometric sensor of the amyloid fibrils. Detailed photophysical properties of 2Me-DABT in amyloid fibrils and different solvent media were studied to understand its sensory activity. Fluorescence resonance energy transfer (FRET) studies suggested that the sites of localization for ThT and 2Me-DABT in amyloid fibrils are not same and their average distance of separation in amyloid fibrils was determined. The experimental data was nicely supported by molecular docking studies, which confirmed the binding of 2Me-DABT in the inner core of the amyloid fibrils.

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Section: Resultsmentioning

confidence: 99%

Benzothiazole‐Based Neutral Ratiometric Fluorescence Sensor for Amyloid Fibrils

Mora

Murudkar

Alamelu

et al. 2016

Chemistry A European J

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“…A combination of molecular dynamics and site-specific mutation studies using b-sheet forming peptides revealed that ThT binds to the surface ridges of the b-sheets, interacting particularly with hydrophobic residues across !4 b-strands ( Figure 6, left panel). These tight hydrophobic interactions hinder inter-ring rotation and LE to TICT state conversion of ThT [32,33,98,99]. Tight binding may also stabilize ThT's positive charge in a twisted conformation.…”

Section: Tht Binding To Amyloid Fibrilsmentioning

confidence: 99%

“…However, this type of binding is the minority option. The major binding mode is in fact via electrostatic interactions with the positive charge on ThT ( Figure 6, right panel), which allows formation of the TICT state with consequent low fluorescence yields [99]. The existence of more than one type of ThT binding site is a salutary reminder that a fibril's change in ThT fluorescence intensity does not necessarily mean a change in the fibril concentration, but could simply arise from a shift in the equilibrium between two ThT binding sites on a given fibril surface.…”

Section: Tht Binding To Amyloid Fibrilsmentioning

confidence: 99%

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

Malmos

Blancas‐Mejia

Weber

et al. 2017

Amyloid

322

243

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Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concerns have recently been raised about its use as a probe specific for amyloid, ThT still continues to be a very valuable tool for studying kinetic aspects of fibrillation and associated inhibition mechanisms. This review aims to provide a conceptual instruction manual, covering appropriate considerations and pitfalls related to the use of ThT. We start by giving a brief introduction to amyloid formation with focus on the morphology of different aggregate species, followed by a discussion of the quality of protein needed to obtain reliable fibrillation data. After an overview of the photochemical basis for ThT's amyloid binding properties and artifacts that may arise from this, we describe how to plan and analyze ThT assays. We conclude with recommendations for complementary techniques to address shortcomings in the ThT assay.

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“…According to our spectrofluorometric analysis, free ThT molecules exhibited a weak fluorescence between 460 and 550 nm upon excitation at 420 nm (Figure c), which is attributed to the rapid quenching of the excited state by nonradiative conformational relaxation in a free form. In contrast, we observed a strong fluorescence from the ThT‐bound insulin nanofibrils; the enhanced ThT fluorescence should be caused by the restriction of conformational relaxation around the central CC bond in its excited electronic state . The fluorescence intensity of ThT increased with the increasing length of insulin fibrils (Figure S4, Supporting Information).…”

Section: Methodsmentioning

confidence: 99%

Thioflavin T‐Amyloid Hybrid Nanostructure for Biocatalytic Photosynthesis

Son

Lee

Wang

et al. 2018

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Amyloidogenic peptides can self-assemble into highly ordered nanostructures consisting of cross β-sheet-rich networks that exhibit unique physicochemical properties and high stability. Light-harvesting amyloid nanofibrils are constructed by employing insulin as a building block and thioflavin T (ThT) as a amyloid-specific photosensitizer. The ability of the self-assembled amyloid scaffold to accommodate and align ThT in high density on its surface allows for efficient energy transfer from the chromophores to the catalytic units in a similar way to natural photosystems. Insulin nanofibrils significantly enhance the photoactivity of ThT by inhibiting nonradiative conformational relaxation around the central CC bonds and narrowing the distance between ThT molecules that are bound to the β-sheet-rich amyloid structure. It is demonstrated that the ThT-amyloid hybrid nanostructure is suitable for biocatalytic solar-to-chemical conversion by integrating the light-harvesting amyloid module (for nicotinamide cofactor regeneration) with a redox biocatalytic module (for enzymatic reduction).

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Ultrafast fluorescence spectroscopy reveals a dominant weakly-emissive population of fibril bound thioflavin-T

Cited by 47 publications

References 26 publications

Benzothiazole‐Based Neutral Ratiometric Fluorescence Sensor for Amyloid Fibrils

Benzothiazole‐Based Neutral Ratiometric Fluorescence Sensor for Amyloid Fibrils

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

Thioflavin T‐Amyloid Hybrid Nanostructure for Biocatalytic Photosynthesis

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