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FRET studies of various conformational states adopted by transthyretin

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Abstract

Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. Here, we have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that we have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of β-strand C, whose degree of unfolding is debated. We found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10–Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating non-native interactions that reduce the Cys10–Trp41 spatial distance, revealing a third distinct conformational state. Overall, our results clarify a negligible degree of unfolding of β-strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states.

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Abbreviations

FRET:

Fluorescence resonance energy transfer

TTR:

Transthyretin

M-TTR:

Monomeric variant of TTR

RBP:

Retinol binding protein

CSF:

Cerebrospinal fluid

SSA:

Senile systemic amyloidosis

FAP:

Familial amyloid polyneuropathy

FAC:

Familial amyloid cardiomyopathy

DMSO:

Dimethyl sulfoxide

GSH:

Glutathione

TCEP:

Tris(2-carboxyethyl)phosphine hydrochloride

TFA:

Trifluoroacetic acid

DACM:

N-(7-Dimethylamino-4-methylcoumarin-3-yl)maleimide

DLS:

Dynamic light scattering

MD:

Molecular dynamics

AU:

Asymmetric unit

CD:

Circular dichroism

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Acknowledgements

We thank the Iranian Ministry of Science Research and Technology for providing the studentship for S.A.G. and the University of Florence for Fondi di Ateneo. F.B.’s research is funded by the Italian MIUR (Programma per Giovani Ricercatori Rita Levi Montalcini 2010). We also thank Martino Bolognesi for insightful discussion. We thank Joel Buxbaum and Xinyi Li for providing the gene coding M-TTR.

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Authors and Affiliations

  1. Dipartimento di Scienze Biomediche Sperimentali e Cliniche “Mario Serio”, Sezione di Scienze Biochimiche, Università degli Studi di Firenze, Viale Morgagni 50, 50134, Florence, Italy

    Seyyed Abolghasem Ghadami, Francesco Bemporad & Fabrizio Chiti

  2. Dipartimento di Bioscienze, Università degli Studi di Milano, 20133, Milan, Italy

    Benedetta Maria Sala & Stefano Ricagno

  3. Center for Complexity and Biosystems, Department of Physics, Università degli Studi di Milano and INFN, via Celoria 16, 20133, Milan, Italy

    Guido Tiana

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  1. Seyyed Abolghasem Ghadami
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  2. Francesco Bemporad
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  3. Benedetta Maria Sala
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Correspondence to Fabrizio Chiti.

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Ghadami, S.A., Bemporad, F., Sala, B.M. et al. FRET studies of various conformational states adopted by transthyretin. Cell. Mol. Life Sci. 74, 3577–3598 (2017). https://doi.org/10.1007/s00018-017-2533-x

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