Advertisement

Cellular and Molecular Life Sciences

, Volume 74, Issue 19, pp 3577–3598 | Cite as

FRET studies of various conformational states adopted by transthyretin

  • Seyyed Abolghasem Ghadami
  • Francesco Bemporad
  • Benedetta Maria Sala
  • Guido Tiana
  • Stefano Ricagno
  • Fabrizio Chiti
Original Article
  • 625 Downloads

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.

Keywords

Protein aggregation Protein misfolding Protein folding Folding intermediate SSA FAP FAC 

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

Notes

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.

References

  1. 1.
    Soprano DR, Herbert J, Soprano KJ, Schon EA, Goodman DS (1985) Demonstration of transthyretin mRNA in the brain and other extrahepatic tissues in the rat. J Biol Chem 260(21):11793–11798PubMedGoogle Scholar
  2. 2.
    Stauder AJ, Dickson PW, Aldred AR, Schreiber G, Mendelsohn FA, Hudson P (1986) Synthesis of transthyretin (pre-albumin) mRNA in choroid plexus epithelial cells, localized by in situ hybridization in rat brain. J Histochem Cytochem 34(7):949–952CrossRefPubMedGoogle Scholar
  3. 3.
    Herbert J, Wilcox JN, Pham KT, Fremeau RT Jr, Zeviani M, Dwork A, Soprano DR, Makover A, Goodman DS, Zimmerman EA et al (1986) Transthyretin: a choroid plexus-specific transport protein in human brain. The 1986 S. Weir Mitchell award. Neurology 36(7):900–911CrossRefPubMedGoogle Scholar
  4. 4.
    Jacobsson B (1989) Localization of transthyretin-mRNA and of immunoreactive transthyretin in the human fetus. Virchows Arch A Pathol Anat Histopathol 415(3):259–263CrossRefPubMedGoogle Scholar
  5. 5.
    Jacobsson B, Collins VP, Grimelius L, Pettersson T, Sandstedt B, Carlstrom A (1989) Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic islets. J Histochem Cytochem 37(1):31–37CrossRefPubMedGoogle Scholar
  6. 6.
    Murakami T, Ohsawa Y, Zhenghua L, Yamamura K, Sunada Y (2010) The transthyretin gene is expressed in Schwann cells of peripheral nerves. Brain Res 1348:222–225. doi: 10.1016/j.brainres.2010.06.017 CrossRefPubMedGoogle Scholar
  7. 7.
    Wakasugi S, Maeda S, Shimada K (1986) Structure and expression of the mouse prealbumin gene. J Biochem 100(1):49–58CrossRefPubMedGoogle Scholar
  8. 8.
    Buxbaum JN, Reixach N (2009) Transthyretin: the servant of many masters. Cell Mol Life Sci 66(19):3095–3101. doi: 10.1007/s00018-009-0109-0 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Reixach N, Foss TR, Santelli E, Pascual J, Kelly JW, Buxbaum JN (2008) Human-murine transthyretin heterotetramers are kinetically stable and non-amyloidogenic. A lesson in the generation of transgenic models of diseases involving oligomeric proteins. J Biol Chem 283(4):2098–2107. doi: 10.1074/jbc.M708028200 CrossRefPubMedGoogle Scholar
  10. 10.
    Sekijima Y (2015) Transthyretin (ATTR) amyloidosis: clinical spectrum, molecular pathogenesis and disease-modifying treatments. J Neurol Neurosurg Psychiatry 86(9):1036–1043. doi: 10.1136/jnnp-2014-308724 CrossRefPubMedGoogle Scholar
  11. 11.
    Sipe JD, Benson MD, Buxbaum JN, Ikeda SI, Merlini G, Saraiva MJ, Westermark P (2016) Amyloid fibril proteins and amyloidosis: chemical identification and clinical classification International Society of Amyloidosis 2016 Nomenclature Guidelines. Amyloid 23(4):209–213. doi: 10.1080/13506129.2016.1257986 CrossRefPubMedGoogle Scholar
  12. 12.
    Lie JT, Hammond PI (1988) Pathology of the senescent heart: anatomic observations on 237 autopsy studies of patients 90 to 105 years old. Mayo Clin Proc 63(6):552–564CrossRefPubMedGoogle Scholar
  13. 13.
    Tanskanen M, Peuralinna T, Polvikoski T, Notkola IL, Sulkava R, Hardy J, Singleton A, Kiuru-Enari S, Paetau A, Tienari PJ, Myllykangas L (2008) Senile systemic amyloidosis affects 25% of the very aged and associates with genetic variation in alpha2-macroglobulin and tau: a population-based autopsy study. Ann Med 40(3):232–239. doi: 10.1080/07853890701842988 CrossRefPubMedGoogle Scholar
  14. 14.
    Connors LH, Lim A, Prokaeva T, Roskens VA, Costello CE (2003) Tabulation of human transthyretin (TTR) variants, 2003. Amyloid 10(3):160–184. doi: 10.3109/13506120308998998 CrossRefPubMedGoogle Scholar
  15. 15.
    Garzuly F, Vidal R, Wisniewski T, Brittig F, Budka H (1996) Familial meningocerebrovascular amyloidosis, Hungarian type, with mutant transthyretin (TTR Asp18Gly). Neurology 47(6):1562–1567CrossRefPubMedGoogle Scholar
  16. 16.
    Cornwell GG 3rd, Sletten K, Johansson B, Westermark P (1988) Evidence that the amyloid fibril protein in senile systemic amyloidosis is derived from normal prealbumin. Biochem Biophys Res Commun 154(2):648–653CrossRefPubMedGoogle Scholar
  17. 17.
    Saraiva MJ, Costa PP, Goodman DS (1988) Transthyretin (prealbumin) in familial amyloidotic polyneuropathy: genetic and functional aspects. Adv Neurol 48:189–200PubMedGoogle Scholar
  18. 18.
    Westermark P, Sletten K, Johansson B, Cornwell GG 3rd (1990) Fibril in senile systemic amyloidosis is derived from normal transthyretin. Proc Natl Acad Sci USA 87(7):2843–2845CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Mccutchen SL, Colon W, Kelly JW (1993) Transthyretin mutation Leu-55-Pro significantly alters tetramer stability and increases amyloidogenicity. Biochemistry 32(45):12119–12127. doi: 10.1021/bi00096a024 CrossRefPubMedGoogle Scholar
  20. 20.
    Sekijima Y, Wiseman RL, Matteson J, Hammarstrom P, Miller SR, Sawkar AR, Balch WE, Kelly JW (2005) The biological and chemical basis for tissue-selective amyloid disease. Cell 121(1):73–85. doi: 10.1016/j.cell.2005.01.018 CrossRefPubMedGoogle Scholar
  21. 21.
    Steinrauf LK, Hamilton JA, Braden BC, Murrell JR, Benson MD (1993) X-ray crystal structure of the Ala-109 → Thr variant of human transthyretin which produces euthyroid hyperthyroxinemia. J Biol Chem 268(4):2425–2430PubMedGoogle Scholar
  22. 22.
    Kelly JW, Colon W, Lai ZH, Lashuel HA, McCulloch J, McCutchen SL, Miroy GJ, Peterson SA (1997) Transthyretin quaternary and tertiary structural changes facilitate misassembly into amyloid. Adv Protein Chem 50:161–181. doi: 10.1016/S0065-3233(08)60321-6 CrossRefPubMedGoogle Scholar
  23. 23.
    Lai Z, Colon W, Kelly JW (1996) The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid. Biochemistry 35(20):6470–6482. doi: 10.1021/bi952501g CrossRefPubMedGoogle Scholar
  24. 24.
    Liu K, Cho HS, Hoyt DW, Nguyen TN, Olds P, Kelly JW, Wemmer DE (2000) Deuterium–proton exchange on the native wild-type transthyretin tetramer identifies the stable core of the individual subunits and indicates mobility at the subunit interface. J Mol Biol 303(4):555–565. doi: 10.1006/jmbi.2000.4164 CrossRefPubMedGoogle Scholar
  25. 25.
    Jiang X, Smith CS, Petrassi HM, Hammarstrom P, White JT, Sacchettini JC, Kelly JW (2001) An engineered transthyretin monomer that is nonamyloidogenic, unless it is partially denatured. Biochemistry 40(38):11442–11452CrossRefPubMedGoogle Scholar
  26. 26.
    Lim KH, Dyson HJ, Kelly JW, Wright PE (2013) Localized structural fluctuations promote amyloidogenic conformations in transthyretin. J Mol Biol 425(6):977–988. doi: 10.1016/j.jmb.2013.01.008 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Lim KH, Dasari AK, Hung I, Gan Z, Kelly JW, Wemmer DE (2016) Structural changes associated with transthyretin misfolding and amyloid formation revealed by solution and solid-state NMR. Biochemistry 55(13):1941–1944. doi: 10.1021/acs.biochem.6b00164 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Conti S, Li X, Gianni S, Ghadami SA, Buxbaum J, Cecchi C, Chiti F, Bemporad F (2014) A complex equilibrium among partially unfolded conformations in monomeric transthyretin. Biochemistry 53(27):4381–4392. doi: 10.1021/bi500430w CrossRefPubMedGoogle Scholar
  29. 29.
    Clegg RM (1995) Fluorescence resonance energy transfer. Curr Opin Biotechnol 6(1):103–110CrossRefPubMedGoogle Scholar
  30. 30.
    Leslie AGW (1992) Joint CCP4 and ESF-EACMB newsletter on protein crystallography. SERC Daresbury Laboratory, WarringtonGoogle Scholar
  31. 31.
    Collaborative Computational Project N (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50(Pt 5):760–763. doi: 10.1107/S0907444994003112 CrossRefGoogle Scholar
  32. 32.
    Evans P (2006) Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62(Pt 1):72–82. doi: 10.1107/S0907444905036693 CrossRefPubMedGoogle Scholar
  33. 33.
    McNicholas S, Potterton E, Wilson KS, Noble ME (2011) Presenting your structures: the CCP4mg molecular-graphics software. Acta Crystallogr D Biol Crystallogr 67(Pt 4):386–394. doi: 10.1107/S0907444911007281 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Long F, Vagin AA, Young P, Murshudov GN (2008) BALBES: a molecular-replacement pipeline. Acta Crystallogr D Biol Crystallogr 64(Pt 1):125–132. doi: 10.1107/S0907444907050172 CrossRefPubMedGoogle Scholar
  35. 35.
    Vagin A, Teplyakov A (1997) MOLREP: an automated program for molecular replacement. J Appl Crystallogr 30:1022–1025. doi: 10.1107/S0021889897006766 CrossRefGoogle Scholar
  36. 36.
    Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221. doi: 10.1107/S0907444909052925 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53(Pt 3):240–255. doi: 10.1107/S0907444996012255 CrossRefPubMedGoogle Scholar
  38. 38.
    Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126–2132. doi: 10.1107/S0907444904019158 CrossRefPubMedGoogle Scholar
  39. 39.
    Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C (2006) Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65(3):712–725. doi: 10.1002/prot.21123 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general amber force field. J Comput Chem 25(9):1157–1174. doi: 10.1002/jcc.20035 CrossRefPubMedGoogle Scholar
  41. 41.
    Santoro MM, Bolen DW (1988) Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27(21):8063–8068CrossRefPubMedGoogle Scholar
  42. 42.
    Pires RH, Karsai A, Saraiva MJ, Damas AM, Kellermayer MS (2012) Distinct annular oligomers captured along the assembly and disassembly pathways of transthyretin amyloid protofibrils. PLoS One 7(9):e44992. doi: 10.1371/journal.pone.0044992 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Peterson SA, Klabunde T, Lashuel HA, Purkey H, Sacchettini JC, Kelly JW (1998) Inhibiting transthyretin conformational changes that lead to amyloid fibril formation. Proc Natl Acad Sci USA 95(22):12956–12960CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New YorkCrossRefGoogle Scholar
  45. 45.
    Nazarov PV, Koehorst RB, Vos WL, Apanasovich VV, Hemminga MA (2006) FRET study of membrane proteins: simulation-based fitting for analysis of membrane protein embedment and association. Biophys J 91(2):454–466. doi: 10.1529/biophysj.106.082867 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Visser NV, Westphal AH, van Hoek A, van Mierlo CP, Visser AJ, van Amerongen H (2008) Tryptophan-tryptophan energy migration as a tool to follow apoflavodoxin folding. Biophys J 95(5):2462–2469. doi: 10.1529/biophysj.108.132001 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Hurshman AR, White JT, Powers ET, Kelly JW (2004) Transthyretin aggregation under partially denaturing conditions is a downhill polymerization. Biochemistry 43(23):7365–7381. doi: 10.1021/bi049621l CrossRefPubMedGoogle Scholar
  48. 48.
    Cascella R, Conti S, Mannini B, Li X, Buxbaum JN, Tiribilli B, Chiti F, Cecchi C (2013) Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro. Biochim Biophys Acta 1832 12:2302–2314. doi: 10.1016/j.bbadis.2013.09.011 CrossRefGoogle Scholar
  49. 49.
    Cappelli S, Penco A, Mannini B, Cascella R, Wilson MR, Ecroyd H, Li X, Buxbaum JN, Dobson CM, Cecchi C, Relini A, Chiti F (2016) Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations. Biol Chem 397(5):401–415. doi: 10.1515/hsz-2015-0250 CrossRefPubMedGoogle Scholar
  50. 50.
    Das JK, Mall SS, Bej A, Mukherjee S (2014) Conformational flexibility tunes the propensity of transthyretin to form fibrils through non-native intermediate states. Angew Chem Int Ed Engl 53(47):12781–12784. doi: 10.1002/anie.201407323 CrossRefPubMedGoogle Scholar
  51. 51.
    Yazaki M, Connors LH, Eagle RC Jr, Leff SR, Skinner M, Benson MD (2002) Transthyretin amyloidosis associated with a novel variant (Trp41Leu) presenting with vitreous opacities. Amyloid 9(4):263–267CrossRefPubMedGoogle Scholar
  52. 52.
    Yazaki M, Varga J, Dyck PJ, Benson MD (2002) A new transthyretin variant Leu55Gln in a patient with systemic amyloidosis. Amyloid 9(4):268–271CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing 2017

Authors and Affiliations

  • Seyyed Abolghasem Ghadami
    • 1
  • Francesco Bemporad
    • 1
  • Benedetta Maria Sala
    • 2
  • Guido Tiana
    • 3
  • Stefano Ricagno
    • 2
  • Fabrizio Chiti
    • 1
  1. 1.Dipartimento di Scienze Biomediche Sperimentali e Cliniche “Mario Serio”, Sezione di Scienze BiochimicheUniversità degli Studi di FirenzeFlorenceItaly
  2. 2.Dipartimento di BioscienzeUniversità degli Studi di MilanoMilanItaly
  3. 3.Center for Complexity and Biosystems, Department of PhysicsUniversità degli Studi di Milano and INFNMilanItaly

Personalised recommendations