title: Phosphorylation as conformational switch from the native to amyloid state: Trp-cage as a protein aggregation model.
abstract: The 20 residue long Trp-cage miniprotein is an excellent model for both computational and experimental studies of protein folding and stability. Recently, great attention emerged to study disease-related protein misfolding, aggregation, and amyloid formation, with the aim of revealing their structural and thermodynamic background. Trp-cage is sensitive to both environmental and structure-modifying effects. It aggregates with ease upon structure destabilization, and thus it is suitable for modeling aggregation and amyloid formation. Here, we characterize the amyloid formation of several sequence modified and side-chain phosphorylated Trp-cage variants. We applied NMR, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies, molecular dynamics (MD) simulations, and transmission electron microscopy (TEM) in conjunction with thioflavin-T (ThT) fluorescence measurements to reveal the structural consequences of side-chain phosphorylation. We demonstrate that the native fold is destabilized upon serine phosphorylation, and the resultant highly dynamic structures form amyloid-like ordered aggregates with high intermolecular β-structure content. The only exception is the D9S(P) variant, which follows an alternative aggregation process by forming thin fibrils, presenting a CD spectrum of PPII helix, and showing low ThT binding capability. We propose a complex aggregation model for these Trp-cage miniproteins. This model assumes an additional aggregated state, a collagen triple helical form that can precede amyloid formation. The phosphorylation of a single serine residue serves as a conformational switch, triggering aggregation, otherwise mediated by kinases in cell. We show that Trp-cage miniprotein is indeed a realistic model of larger globular systems of composite folding and aggregation landscapes and helps us to understand the fundamentals of deleterious protein aggregation and amyloid formation.
authors.affiliation: Department of Biochemistry, ‡MTA-ELTE NAP B Neuroimmunology Research Group, and §Department of Anatomy, Cell and Developmental Biology, Institute of Biology Eötvös Loránd University , Pázmány P. sétány 1/C, Budapest, H-1117 Hungary.
authors.forename: József
authors.initials: J
authors.lastname: Kardos
authors.forename: Bence
authors.initials: B
authors.lastname: Kiss
authors.forename: András
authors.initials: A
authors.lastname: Micsonai
authors.forename: Petra
authors.initials: P
authors.lastname: Rovó
authors.forename: Dóra K
authors.initials: DK
authors.lastname: Menyhárd
authors.forename: János
authors.initials: J
authors.lastname: Kovács
authors.forename: Györgyi
authors.initials: G
authors.lastname: Váradi
authors.forename: Gábor K
authors.initials: GK
authors.lastname: Tóth
authors.forename: András
authors.initials: A
authors.lastname: Perczel
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry*; D052160:Benzothiazoles; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy; D046529:Microscopy, Electron, Transmission; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D017550:Spectroscopy, Fourier Transform Infrared; D013844:Thiazoles / Q000737:chemistry
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D010455:Peptides; D013844:Thiazoles; C508725:Trp-cage peptide; C009462:thioflavin T