Amyloid-like Fibril Formation in an All β-Barrel Protein

Abstract

Acidic fibroblast growth factor from newt (Notopthalmus viridescens) is a ∼15-kDa, all β-sheet protein devoid of disulfide bonds. In the present study, we investigate the effects of 2,2,2-trifluoroethanol (TFE) on the structure of newt acidic fibroblast growth factor (nFGF-1). The protein aggregates maximally in 10% (v/v) TFE. Congo red and thioflavin T binding experiments suggest that the aggregates induced by TFE have properties resembling the amyloid fibrils. Transmission electron microscopy and x-ray fiber diffraction data show that the fibrils (induced by TFE) are straight, unbranched, and have a cross-β structure with an average diameter of 10–15 Å. Preformed fibrils (induced by TFE) of nFGF-1 are observed to seed amyloid-like fibril formation in solutions containing the protein (nFGF-1) in the native β-barrel conformation. Fluorescence, far-UV CD, anilino-8-napthalene sulfonate binding, multidimensional NMR, and Fourier transformed infrared spectroscopy data reveal that formation of a partially structured intermediate state(s) precedes the onset of the fibrillation process. The native β-barrel structure of nFGF-1 appears to be disrupted in the partially structured intermediate state(s). The protein in the partially structured intermediate state(s) is found to be “sticky” with a solvent-exposed non-polar surface(s). Amyloid fibril formation appears to occur due to coalescence of the protein in the partially structured intermediate state(s) through solvent-exposed non-polar surfaces and intermolecular β-sheet formation among the extended, linear β-strands in the protein. Acidic fibroblast growth factor from newt (Notopthalmus viridescens) is a ∼15-kDa, all β-sheet protein devoid of disulfide bonds. In the present study, we investigate the effects of 2,2,2-trifluoroethanol (TFE) on the structure of newt acidic fibroblast growth factor (nFGF-1). The protein aggregates maximally in 10% (v/v) TFE. Congo red and thioflavin T binding experiments suggest that the aggregates induced by TFE have properties resembling the amyloid fibrils. Transmission electron microscopy and x-ray fiber diffraction data show that the fibrils (induced by TFE) are straight, unbranched, and have a cross-β structure with an average diameter of 10–15 Å. Preformed fibrils (induced by TFE) of nFGF-1 are observed to seed amyloid-like fibril formation in solutions containing the protein (nFGF-1) in the native β-barrel conformation. Fluorescence, far-UV CD, anilino-8-napthalene sulfonate binding, multidimensional NMR, and Fourier transformed infrared spectroscopy data reveal that formation of a partially structured intermediate state(s) precedes the onset of the fibrillation process. The native β-barrel structure of nFGF-1 appears to be disrupted in the partially structured intermediate state(s). The protein in the partially structured intermediate state(s) is found to be “sticky” with a solvent-exposed non-polar surface(s). Amyloid fibril formation appears to occur due to coalescence of the protein in the partially structured intermediate state(s) through solvent-exposed non-polar surfaces and intermolecular β-sheet formation among the extended, linear β-strands in the protein. 2,2,2-trifluoroethanol newt acidic fibroblast growth factor thioflavin T 1-anilino-8-napthalene sulfonate heteronuclear single quantum coherence Fourier transform infrared spectroscopy Protein aggregation is a problem of importance not only in biotechnology but also in health-related industries (1Chan W. Helms L.R. Brooks I. Lee G. Ngola S. McNulty D. Maleeff B. Hensley P. Wetzel R. Fold. Des. 1996; 1: 77-89Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 2Klein J. 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J. 2000; PubMed Scopus Google Scholar). TFE is observed to amyloid-like fibrils in The formation of amyloid-like fibrils is by the of a partially structured intermediate state(s) in the of from and from sulfonate thioflavin T and Congo red from of all solutions in containing experiments nFGF-1 from transformed cells. The nFGF-1 of the and The protein on a a of the protein by an The of the protein The of the form of nFGF-1 by the form of nFGF-1 to the of by the containing the with the an to protein of in containing The The into a and with containing the of the to a nFGF-1 with containing The of the nFGF-1 by The of the by Protein on the of the protein be that the newt we as is in all the experiments in the present the of the by the of a of The E. is a and the with Protein in the of of the and to nFGF-1 the as in the The of by on a of of the protein (nFGF-1) in appropriate of TFE. The of the protein in the experiments The of the solutions by The of the on a experiments a and the of an average of and an average of to the The of the protein The and the in the by the The of protein in experiments Protein in of TFE the red thioflavin The solutions the of the of on a a with a of The and and and the Congo red binding of the containing of a solution of the on a in the of of amyloid fibrils in TFE) of nFGF-1 on a electron of the protein a of to and The fibrils by the of of by with The fibrils by the of of and with The induced in (v/v) TFE the of and to The x-ray of fibrils a on an of the The on a to the x-ray fibrils. The by the of nFGF-1 in (v/v) TFE to The in containing and the of solution of the protein to solution induced of the protein. The experiments with appropriate The the of an solution of nFGF-1 in the of the of the and the the of an of the solution into The by the in the In the solution by electron microscopy to the of fibrils and their experiments on a a protein of an of sulfonate binding experiments on nFGF-1 of TFE an of The and The of the and the protein (nFGF-1) and experiments on with a to all heteronuclear single quantum coherence the data in the data in The all of TFE. The of the protein in and and the of on a and The the as Protein aggregates formed in (v/v) TFE a protein of a and the The in a The of the aggregates in a by a of nFGF-1 (v/v) and (v/v) TFE by the protein in containing appropriate of with a of and and a Fourier as to the of the in the and nFGF-1 is a ∼15-kDa, all β-sheet protein with disulfide S. H.M. Protein Sci. 2002; 11: PubMed Scopus Google Scholar). The protein and the structural β-strands into a β-barrel The of to investigate the of the of In we the structural in nFGF-1 of TFE far-UV The far-UV of nFGF-1 and an characteristic of type II β-barrel proteins The is believed to the of the β-strands in the β-barrel of the protein. in the of the a TFE (v/v) a TFE of the far-UV of the protein show The is the of the native β-barrel The of the is by the of a in TFE (v/v) the formation of β-sheet conformation. The of the is observed to with the in the TFE from (v/v) to (v/v) of TFE the far-UV of the protein show and the of nFGF-1 in native the induced of TFE appears to be in The of from the far-UV of the protein is The of the of the protein Biochem. PubMed Scopus Google Scholar, N. 2000; PubMed Scopus Google Scholar) (v/v) TFE) is to be The suggest that the induced by TFE in nFGF-1 occur in In the the β-barrel is in the formation of β-sheet conformation. In the of the of the protein form conformation. The protein with TFE in the of 10% (v/v) to (v/v) and are observed to the aggregation of the protein (nFGF-1) is by the in the of TFE of of the protein in of the aggregation that the protein to aggregate maximally and (v/v) the protein is observed to in an β-sheet The of aggregates formed appears to with the in the formation of the β-sheet in the protein. In the aggregates formed TFE 10% to (v/v) TFE) are and of TFE (v/v) the protein solution of suggest that the aggregation of nFGF-1 is to the of structural induced in the protein of β-sheet appears to and of with the of protein by Dobson and on in the protein F. Taddei N. Baroni F. Stefani M. Ramponi G. Dobson C.M. Biol. 2002; 9: PubMed Scopus Google Scholar, N. Chiti F. Stefani M. Dobson C.M. Ramponi G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). aggregation and of proteins Protein aggregation to the β-sheet of the of the protein in the F. Taddei N. Baroni F. Stefani M. Ramponi G. Dobson C.M. Biol. 2002; 9: PubMed Scopus Google Scholar). In that to the aggregation and that the the aggregation F. Taddei N. Baroni F. Stefani M. Ramponi G. Dobson C.M. Biol. 2002; 9: PubMed Scopus Google Scholar). The aggregates of nFGF-1 formed in TFE (v/v) TFE) have a that properties characteristic of amyloid fibrils. by the and properties of the Congo red is a to amyloid fibril formation by proteins N. J. PubMed Scopus Google Scholar). The of the is to a red binding to β-sheet in the amyloid fibrils D.A. J. Biol. 2000; PubMed Scopus Google Scholar). the aggregation is 10% Congo red binding is observed maximally (v/v) TFE The binding is not only by a in the but also by a red in the to (v/v) TFE the of the with the in of the suggest that the fibrils formed maximally (v/v) TFE have amyloid-like The of the aggregates formed also is a as a to amyloid fibril formation M. Blake Chem. 1997; PubMed Google Scholar). The of is to binding to the linear of β-strands in the amyloid fibrils. is in the of the TFE 10% (v/v) an in the is observed the to the protein in TFE that amyloid-like fibrils of nFGF-1 maximally in (v/v) TFE. The of the aggregates formed in (v/v) TFE by electron microscopy and x-ray fiber reveal that the aggregates formed are and unbranched, with an average diameter of 10–15 and is to the of the fibrils formed from proteins M. Blake Chem. 1997; PubMed Google Scholar). fiber diffraction of the nFGF-1 aggregates a on the and of fibrils with a cross-β structure The from the the β-sheet is and The is to the is the of the electron microscopy and x-ray fiber diffraction experiments that the aggregates formed in (v/v) TFE features resembling that of the amyloid fibrils. is a in aggregation and as it is in J. Scholar). that of fibrils formed by it is to solutions the of formation of amyloid-like fibrils J.D. Lansbury P.T. Annu. Rev. Biochem. 1997; 66: 355-407Crossref Scopus (1413) Google Scholar). Recently, Bucciantini et al. (27Bucciantini M. Giannoni E. Chiti F. Baroni F. Formgli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2154) Google Scholar) that amyloid fibrils in of proteins including SH3 domain from phosphatidylinositol and the domain of the E. are not with are highly cytotoxic and induced amyloid-like fibrils in cultured NIH3T3 fibroblast cells (27Bucciantini M. Giannoni E. Chiti F. Baroni F. Formgli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2154) Google Scholar). In we the of the amyloid-like fibrils of nFGF-1 induced in TFE. The of the fibrils of nFGF-1 (induced in TFE to seed fibril formation by the in the the of of fibrils into solutions of native be observed from that the the of the fibrils (induced in experiments by the of of nFGF-1 fibrils into a of native nFGF-1 solution that the of aggregation with the in the of the fibrils the of induced aggregation from the appears to be of the of the fibrils be that with an of the fibrils into the containing and a solution of nFGF-1 with the to a protein of under the show of in aggregation observed from the experiments that fibrils of nFGF-1 in TFE) are of fibril formation in solutions containing the protein (nFGF-1) in the native conformation. binding experiments to the aggregates by the fibrils have amyloid-like in the of the be observed of the native protein with fibrils suggest that the induced aggregates properties resembling the amyloid fibrils. In electron that the induced aggregates are with an average diameter of S. D. and In the of the experiments that fibril growth is by a mechanism to that observed in growth and process. in also observed in studies of human lysozyme and Zurdo J. A. W. Pepys M. V. Dobson C.M. J. Biol. 2000; PubMed Scopus Google Scholar). Recently, fibrils generated under in vitro conditions in lysozyme is not with amyloid have to seed fibril formation of protein in the native Zurdo J. Robinson C.V. Dobson C.M. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). vitro studies with the with Alzheimer's suggest that of the onset of amyloid and of by a mechanism J.D. Lansbury P.T. Annu. Rev. Biochem. 1997; 66: 355-407Crossref Scopus (1413) Google Scholar). Recently, is in the molecular to the formation of amyloid fibrils. on in it is now increasingly believed that amyloid fibril formation the of partially that are of fibrils C.E. Dobson C.M. J. Am. Chem. Soc. 2000; 122: 12707-12713Crossref Scopus (149) Google Scholar, 23Pertinhez T.A. Bouchard M. Tomlinson E.J. Wain R. Ferguson S.J. 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Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. G. J. Biol. Chem. Full Text PDF Google Scholar). is to the of and in the of the of the S. H.M. Protein Sci. 2002; 11: PubMed Scopus Google Scholar). the is of the a with an the of to the the of the protein J.M. G. J. Biol. Chem. Full Text PDF Google Scholar). The of nFGF-1 not of TFE (v/v) in the TFE of to (v/v) the of the to of structural in the protein The effects on the protein are (v/v) TFE. the data in with that far-UV β-sheet in the that nFGF-1 in a partially structured with and of TFE (v/v) the show a in the a in the of the of in the protein. is a that is to to solvent-exposed surfaces in a characteristic of the formation of partially structured state(s) PubMed Scopus Google Scholar). The binding to the native and state(s) of protein J.M. G. J. Biol. Chem. Full Text PDF Google Scholar). The binding of to the protein not (v/v) TFE TFE (v/v) the of the a (v/v) TFE. The of the binding to the protein in (v/v) TFE is that observed it is to the native of the protein In the binding to the protein in (v/v) TFE a of (from to in the of that a partially structured state(s) with a solvent-exposed non-polar (v/v) TFE) the formation of amyloid-like fibrils. (v/v) the of the in the of the a in the of the partially structured intermediate binding to state(s) appears that the partially structured intermediate is maximally (v/v) is and a to aggregate through solvent-exposed and intermolecular β-sheet formation be The aggregates to form organized amyloid-like fibrils. spectroscopy the the of of proteins Dobson C.M. Biol. 1997; Scopus Google Scholar). experiments have to be of Dobson C.M. Biol. 1997; Scopus Google Scholar). the of to investigate the induced by TFE In as a of the of a protein Dobson C.M. Biol. 1997; Scopus Google Scholar). The of nFGF-1 in native TFE) is and all the in the be S. H.M. Protein Sci. 2002; 11: PubMed Scopus Google Scholar). be observed in the of the protein a TFE of (v/v) not the structural features of the partially structured intermediate state(s) (v/v) TFE the protein aggregates (v/v) the structural of the partially structured intermediate state(s) from the observed in the (v/v) TFE. of the protein in (v/v) TFE that the features are of to the protein. to in the structure show in and that the and of the nFGF-1 through also show S. H.M. Protein Sci. 2002; 11: PubMed Scopus Google Scholar, that the native β-barrel is disrupted in the partially structured intermediate state(s) that fibril the structural features of the protein in (v/v) TFE and (v/v) TFE the partially structured state(s) is maximally be it not be to that the of the protein in the partially structured intermediate state(s) (v/v) TFE) be not to that observed in (v/v) TFE. The of the protein in (v/v) TFE that the of the is that of the protein are in a The far-UV and data in (v/v) TFE) in that the protein as a with only of the polypeptide in conformation. be that in the of the of the in (v/v) TFE be the of formed in the protein in (v/v) TFE. are to a the amyloid-like fibrils in is that such as TFE effects by partially the and of proteins R. P. J. Protein 1996; PubMed Scopus Google Scholar, 1997; Scopus Google Scholar). The effects of TFE on proteins from their the that the native of proteins Protein Sci. 1993; PubMed Scopus Google Scholar). In the induced by TFE 10% appears to be the of the the native β-barrel structure to the formation of a partially structured intermediate state(s) The of non-polar in the partially structured state(s) in (v/v) TFE) is from the binding of the protein in the intermediate state(s) to the of the native β-barrel is in the of of the in the in TFE and the of the with the of the The of the native β-barrel is also from the in (v/v) the and the β-barrel structure of the protein of the protein in the TFE of to is by the coalescence of the β-sheet in the partially structured intermediate state(s) The amyloid-like fibril formation appears to due to the and of the β-sheet through intermolecular of intermolecular is by the of the in the of the amyloid-like fibril J.D. Lansbury P.T. Annu. Rev. Biochem. 1997; 66: 355-407Crossref Scopus (1413) Google Scholar) In to the intermolecular that the of the non-polar in the protein the β-sheet also appears to a the of the polypeptide to form a such as the amyloid-like fibrils The of aggregation observed in of TFE (v/v) TFE) is due to of the the intermolecular β-sheet in the aggregates 1997; Scopus Google Scholar, Protein Sci. 1993; PubMed Scopus Google Scholar). The of non-polar of TFE) appears to the formation of intermolecular the and formation of conformation. suggest that protein aggregation is to the S. J. Am. Chem. Soc. 2002; PubMed Scopus Google Scholar, V.N. J.A. Fink A.L. 2000; 41: PubMed Scopus Google Scholar). of β-sheet appears to aggregation to form and formation of in the appears to Therefore, the of to protein aggregation in vivo and in In TFE is not a the β-sheet to structural induced by TFE be as a to the of the protein from the form to the β-sheet to amyloid fibril of nFGF-1 in native TFE) and the amyloid-like fibril TFE) be observed that the and the native β-barrel structure in the amyloid-like fibril to a the formation of a and are of the intermolecular β-sheet in the amyloid-like In the of the present the of Dobson and (21Fandrich M. Fletcher M.A. Dobson C.M. Nature. 2001; 410: 165-166Crossref PubMed Scopus (731) Google Scholar) that formation of amyloid fibrils is a generic of and proteins and not to the observed in TFE is not a the mechanism of amyloid formation in proteins as into the fibrillation process. the also and