title: Tryptophanyl substitutions in apomyoglobin determine protein aggregation and amyloid-like fibril formation at physiological pH.
abstract: Myoglobin is an alpha-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 beta-structures. In conclusion, under physiological pH conditions, some mutations that affect folding may cause protein aggregation and the formation of amyloid-like fibrils.
authors.affiliation: Dipartimento di Biochimica e Biofisica, Seconda Università degli Studi di Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy.
authors.forename: Ivana
authors.initials: I
authors.lastname: Sirangelo
authors.forename: Clorinda
authors.initials: C
authors.lastname: Malmo
authors.forename: Mariateresa
authors.initials: M
authors.lastname: Casillo
authors.forename: Antonio
authors.initials: A
authors.lastname: Mezzogiorno
authors.forename: Michele
authors.initials: M
authors.lastname: Papa
authors.forename: Gaetano
authors.initials: G
authors.lastname: Irace
mesh_terms: D000682:Amyloid / Q000378:metabolism*; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000682:Amyloid; D001059:Apoproteins; D017931:DNA Primers; D009211:Myoglobin; C010857:apomyoglobin; D014364:Tryptophan