title: Amyloid fibril formation and disaggregation of fragment 1-29 of apomyoglobin: insights into the effect of pH on protein fibrillogenesis.
abstract: The N-terminal fragment 1-29 of horse heart apomyoglobin (apoMb(1-29)) is highly prone to form amyloid-like fibrils at low pH. Fibrillogenesis at pH 2.0 occurs following a nucleation-dependent growth mechanism, as evidenced by the thioflavin T (ThT) assay. Transmission electron microscopy (TEM) confirms the presence of regular amyloid-like fibrils and far-UV circular dichroism (CD) spectra indicate the acquisition of a high content of beta-sheet structure. ThT assay, TEM and CD highlight fast and complete disaggregation of the fibrils, if the pH of a suspension of mature fibrils is increased to 8.3. It is of interest that amyloid-like fibrils form again if the pH of the solution is brought back to 2.0. While apoMb(1-29) fibrils obtained at pH 2.0 are resistant to proteolysis by pepsin, the disaggregated fibrils are easily cleaved at pH 8.3 by trypsin and V8 protease, and some of the resulting fragments aggregate very quickly in the proteolysis mixture, forming amyloid-like fibrils. We show that the increase of amyloidogenicity of apoMb(1-29) following acidification or proteolysis at pH 8.3 can be attributed to the decrease of the peptide net charge following these alterations. The results observed here for apoMb(1-29) provide an experimental basis for explaining the effect of charge and pH on amyloid fibril formation by both unfolded and folded protein systems.
authors.affiliation: CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Picotti
authors.forename: Giorgia
authors.initials: G
authors.lastname: De Franceschi
authors.forename: Erica
authors.initials: E
authors.lastname: Frare
authors.forename: Barbara
authors.initials: B
authors.lastname: Spolaore
authors.forename: Marcello
authors.initials: M
authors.lastname: Zambonin
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Chiti
authors.forename: Patrizia Polverino
authors.initials: PP
authors.lastname: de Laureto
authors.forename: Angelo
authors.initials: A
authors.lastname: Fontana
mesh_terms: D000682:Amyloid / Q000096:biosynthesis* / Q000737:chemistry*; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D013057:Spectrum Analysis; D055672:Static Electricity
chemical_list: D000682:Amyloid; D001059:Apoproteins; D009211:Myoglobin; D010446:Peptide Fragments; C010857:apomyoglobin