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

Abstract

Acidic fibroblast growth factor from newt (Notopthalmus viridescens) is a ϳ15-kDa, all ␤-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-␤ 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 ␤-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 ␤-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 ␤-sheet formation among the extended, linear ␤-strands in the protein.Protein aggregation is a problem of importance not only in biotechnology but also in health-related industries (1, 2). Globular proteins in aqueous solution often tend to aggregate under a variety of conditions of concentration, temperature, pH, and ionic strength (3-6). The morphology of aggregates formed varies considerably and ranges from amorphous forms to highly structured fibrils (7-9). Structured fibrils formed in vitro closely resemble the highly organized amyloid fibrils found in association with a variety of human disorders, including Alzheimer's disease, Creutzfeldt-Jakob disease, Huntington's disease, and type II diabetes (10 -19). Several studies show proteins that are apparently unrelated in sequence and, in their native conformation, aggregate into fibrils that have characteristic amyloid-like structural and histological features (16, 20 -26). Recently, Bucciantini et al. (27) demonstrated that amyloid-like fibrils of SH3 domain (from bovine phosphatidylinositol 3-kinase) induced in vitro in 25% (v/v) 2,2,2-trifluoro ethanol (TFE) 1 (under appropriate conditions) are cytotoxic to fibroblast NIH3T3 cells. The amyloid fibrils generated ex vivo are observed to seed fibrillate in cultured NIH3T3 cells (23). Therefore, it is now increasingly believed that amyloid represents a generic form of polypeptide conformation, and all peptides/proteins have the potential to form amyloid-like fibrils under appropriate conditions (21-27).The...

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