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Novel Trans-Stilbene-based Fluorophores as Probes for Spectral Discrimination of Native and Protofibrillar Transthyretin
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
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2016 (English)In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 7, no 7, 924-940 p.Article in journal (Refereed) Published
Abstract [en]

Accumulation of misfolded transthyretin (TTR) as amyloid fibrils causes various human disorders. Native transthyretin is a neurotrophic protein and is a putative extracellular molecular chaperone. Several fluorophores have been shown in vitro to bind selectively to native TTR. Other compounds, such as thioflavin T, bind TTR amyloid fibrils. The probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to both native and fibrillar TTR, becoming highly fluorescent, but with indistinguishable emission spectra for native and fibrillar TTR. Herein we report our efforts to develop a fluorescent small molecule capable of binding both native and misfolded protofibrillar TTR, providing distinguishable emission spectra. We used microwave synthesis for efficient production of a small library of trans-stilbenes and fluorescence spectral screening of their binding properties. We synthesized and tested 22 trans-stilbenes displaying a variety of functional groups. We successfully developed two naphthyl-based trans-stilbenes probes that detect both TTR states at physiological concentrations. The compounds bound with nanomolar to micromolar affinities and displayed distinct emission maxima upon binding native or misfolded protofibrillar TTR (>100 nm difference). The probes were mainly responsive to environment polarity providing evidence for the divergent hydrophobic structure of the binding sites of these protein conformational states. Furthermore, we were able to successfully use one of these probes to quantify the relative amounts of native and protofibrillar TTR in a dynamic equilibrium. In conclusion, we identified two trans-stilbene-based fluorescent probes, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (11) and (E)-4-(2-(naphthalen-2-yl)vinyl)benzene-1,2-diol (14), that bind native and protofibrillar TTR, providing a wide difference in emission maxima allowing conformational discrimination by fluorescence spectroscopy. We expect these novel molecules to serve as important chemical biology research tools in studies of TTR folding and misfolding.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016. Vol. 7, no 7, 924-940 p.
Keyword [en]
transthyretin, amyloid, stilbene, fluorescence, probe, spectrum
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:liu:diva-122842DOI: 10.1021/acschemneuro.6b00062ISI: 000380297500009PubMedID: 27144293OAI: oai:DiVA.org:liu-122842DiVA: diva2:874261
Note

At the time for thesis presentation publication was in status: Manuscript

Funding agencies:The work was supported by Goran Gustafsson's Foundation (PH), The Swedish Research Council (PH), The Linkoping center for systemic neuroscience, LiU-Neuro, (XW), and Sven and Lilly Lawski's foundation (ME).

Available from: 2015-11-26 Created: 2015-11-26 Last updated: 2016-09-19Bibliographically approved
In thesis
1. Studies on molecular aspects of Transthyretin Amyloidosis
Open this publication in new window or tab >>Studies on molecular aspects of Transthyretin Amyloidosis
2015 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are versatile molecules that play a variety of roles in maintaining the human body, e.g. transport of nutrients. Transthyretin (TTR) is a 55 kDa homotetrameric protein found in human plasma and in the cerebrospinal fluid, responsible for the transport of retinol (vitamin A) and T4 (thyroxine). This protein is probably not essential for life, since TTR knockout mice have normal fetal development and lifespan. TTR, like 25 other human proteins, has been associated to the deposition of amyloid aggregates. Previous research has shown that mutations considerably increase the propensity of the protein to form aggregates. However, the wild type protein also exhibits this ability to aggregate, giving rise to the senile systemic amyloidosis disease that affects 20% people over 80 years of age. It is well accepted that self-association of monomeric subunits triggers the disease through tetramer dissociation, since stabilization of the quaternary structure suppresses aggregate formation.

However, a detailed description of the self-assembly mechanism and fibril structure remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state.

In our efforts to study both native and protofibrillar TTR, we realized the need for development of a fluorescent small molecule capable of binding native and protofibrillar TTR, providing distinguishable emission spectra. We used microwave heating for efficient synthesis and fluorescence spectral screening of compounds. We synthesized and tested 22 analogs displaying a variety of functional groups, most of them linked to a stilbene scaffold. We successfully developed two compounds that detect both TTR states at physiological concentrations. The compounds bound with nM-μM affinities and displayed very distinct emission maxima upon binding native or protofibrillar TTR (> 100 nm difference).

We expect these new findings regarding protofibril self-assembly mechanism, together with our novel molecules serve as important tools in future studies of TTR amyloid formation.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. 37 p.
Series
Linköping Studies in Science and Technology. Thesis, ISSN 0280-7971 ; 1740
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-122843 (URN)978-91-7685-866-0 (print) (ISBN)
Available from: 2015-11-26 Created: 2015-11-26 Last updated: 2015-11-26Bibliographically approved

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