Abstract
Structural changes of globular proteins and their resultant amyloid aggregation have been associated with various human diseases, such as lysozyme amyloidosis and light-chain amyloidosis. Because many globular proteins can convert into amyloid fibrils in vitro, the mechanisms of amyloid fibril formation have been studied in various experimental systems, but several questions remain unresolved. Here, using several approaches, such as turbidimetry, fluorescence and CD spectroscopy, EM, and isothermal titration calorimetry, we examined the binding of polyphosphates to hen egg-white lysozyme under acidic conditions and observed polyphosphate-induced structural changes of the protein promoting its aggregation. Our data indicate that negatively charged polyphosphates bind to protein molecules with a net positive charge. The polyphosphate-bound, structurally destabilized protein molecules then start assembling into insoluble amorphous aggregates once they pass the solubility limit. We further show that the polyphosphates decrease the solubility limit of the protein and near this limit, the protein molecules are in a labile state and highly prone to converting into amyloid fibrils. Our results explain how polyphosphates affect amorphous aggregation of proteins, how amyloid formation is induced in the presence of polyphosphates, and how polyphosphate chain length is an important factor in amyloid formation.
Highlights
Structural changes of globular proteins and their resultant amyloid aggregation have been associated with various human diseases, such as lysozyme amyloidosis and light-chain amyloidosis
We examined the binding of polyphosphates to hen egg-white lysozyme (HEWL) molecules, the resultant structural changes of the protein, and subsequent protein aggregation using turbidimetry, fluorescence, and circular dichroism (CD) spectroscopy, transmission electron microscopy (EM), and isothermal titration calorimetry (ITC)
We investigated the amorphous aggregation and fibrillation of HEWL in the presence of polyphosphates under acidic conditions to understand the relationship between the stability and solubility of the protein and its propensity to form amyloid fibrils
Summary
To assess the ability of negatively charged phosphate groups in a linear chain linked by phosphoanhydride bonds to promote aggregation of positively charged HEWL, we first tested three different sodium phosphates in acidic solution containing 10 mM HCl (pH 2): pyrophosphate (Pyro-P), tripolyphosphate (Tri-P), and tetrapolyphosphate (Tetra-P). As NaCl concentrations increased, the previously turbid solutions became transparent and CD spectra indicated a recovery of protein structure These results indicate that Tetra-P–induced aggregation was accompanied by structural disruption of protein molecules and the original state was restored upon addition of NaCl. Thermal stability of the protein solutions (0.2 mg/ml) containing 10 mM HCl, 1 mM Tetra-P, and NaCl at several concentrations, was examined by CD spectroscopy. The protein solutions at 0.8 and 1.0 M NaCl showed typical thermal denaturation curves These CD data indicate that in the labile state near the solubility limit modulated by Tetra-P and L-PolyP, the protein molecules are structurally destabilized and prone to partially unfolded states, resulting in efficient conversion into amyloid fibrils under ultrasonic irradiation. Each peak in the binding isotherm represents a heat change associated with a single injection, resulting in exothermic
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