Tryptophanyl Substitutions in Apomyoglobin Determine Protein Aggregation and Amyloid-like Fibril Formation at Physiological pH

Abstract

Myoglobin is an ␣-helical globular protein that contains two highly conserved tryptophan residues located at positions 7 and 14 in the N-terminal region of the protein. Replacement of both indole residues with phenylalanine residues, i.e. W7F/W14F, results in the expression of an unstable, not correctly folded protein that does not bind the prosthetic group. Here we report data (Congo red and thioflavine T binding assay, birefringence, and electron microscopy) showing that the double Trp/Phe replacements render apomyoglobin molecules highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions in which most of the wild-type protein is in the native state. In refolding experiments, like the wild-type protein, the W7F/W14F apomyoglobin mutant formed a soluble, partially folded helical state between pH 2.0 and pH 4.0. A pH increase from 4.0 to 7.0 restored the native structure only in the case of the wild-type protein and determined aggregation of W7F/W14F. The circular dichroism spectrum recorded immediately after neutralization showed that the polypeptide consists mainly of ␤-structures. In conclusion, under physiological pH conditions, some mutations that affect folding may cause protein aggregation and the formation of amyloid-like fibrils.Such chronic disorders as Alzheimer's disease, senile systemic amyloidosis, transmissible spongiform encephalopathies, and dialysis-related amyloidosis are characterized by the extracellular deposition of insoluble protein aggregates known as amyloid fibrils (1-6). About 20 proteins are now known to be involved in the generation of amyloid in vivo. Fibril formation is initiated in vitro under conditions that stabilize partially unfolded soluble intermediates of the native proteins either after the partial destabilization of physiologically folded proteins in the case of globular proteins (7) or after the partial stabilization (i.e. folding) of random coil polypeptide chains in the case of natively unfolded proteins (8). Despite substantial differences in both sequence and length (from 40 to 250 residues), all the proteins responsible for amyloid deposition form fibrils composed of ␤-strands oriented perpendicularly to the long axis of the fibril (9). Electron microscopy shows that the fibrils are straight and unbranched and are 40 -120 Å in diameter (9, 10). Also proteins not known to be associated with amyloid disease may form amyloid fibrils under in vitro conditions that favor partially folded states (11-15). These states are more prone to aggregation than the native state because hydrophobic residues, which are largely buried within the core of the native protein, become more exposed upon partial unfolding. The way in which proteins aggregate in the test tube is remarkably similar to how proteins form the so-called "amyloid" deposits. Even myoglobin, an ordinary all-␣ globular protein, can form fibrils containing ␤-strands under experimental conditions that favor the formation of partially folded states (15). Thus, amyloid formation d...

Sonication of proteins causes formation of aggregates that resemble amyloid

“…5; Table 1). These shifts are similar to the characteristic maximum difference at ∼541 nm observed for amyloid (Klunk et al 1999), and the differential absorbance values are comparable to levels observed for other amyloid and amyloid-like fibrils (Gross et al 1999;Chiti et al 2001;Sirangelo et al 2002;KoscielskaKasprzak and Otlewski 2003).…”

“…Protein solutions diluted to 1 mg/mL were incubated in 50 M CR (Chiti et al 2001;Sirangelo et al 2002;Srisailam et al 2002), 20 mM HEPES (pH 7.8) for 20 min. Absorption spectra were acquired using a Cary 1Bio UV/visible spectrophotometer (Varian) at 25°C and a scan rate of 300 nm/min with a 0.3-cm path length cuvette.…”

Fibrillogenesis and Cytotoxic Activity of the Amyloid-forming Apomyoglobin Mutant W7FW14F

“…pH 4.0, the W7FW14F apomyoglobin mutant adopts a soluble molten globule-like conformation similar to that of the wild-type protein (24). In this state, the A, G, and H helices are folded as in the native conformation, whereas the rest of the molecule is essentially unfolded (39 -44).…”

“…Protein Purification-Wild-type and W7FW14F mutant myoglobin were expressed and purified essentially as described elsewhere (24). Proteins were expressed in Escherichia coli BL21 strain as N-terminal His-tagged forms and purified via affinity chromatography on Ni 2ϩ -nitrilotriacetic acid resin (Qiagen).…”

“…We previously demonstrated that the substitution of the two highly conserved tryptophanyl residues of myoglobin, i.e. Trp-7 and Trp-14, with phenylalanine results in the expression of a protein that does not undergo correct folding (23) but aggregates and forms amyloid-like fibrils at physiological pH (24). This mutated protein lends itself to investigations of the relationship between folding and misfolding at molecular level.…”