title: Role of monomer arrangement in the amyloid self-assembly.
abstract: Assembly of amyloid proteins into aggregates requires the ordering of the monomers in oligomers and especially in such highly organized structures as fibrils. This ordering is accompanied by structural transitions leading to the formation of ordered β-structural motifs in proteins and peptides lacking secondary structures. To characterize the effect of the monomer arrangements on the aggregation process at various stages, we performed comparative studies of the yeast prion protein Sup35 heptapeptide (GNNQQNY) along with its dimeric form CGNNQQNY-(d-Pro)-G-GNNQQNY. The (d-Pro)-G linker in this construct is capable of adopting a β-turn, facilitating the assembly of the dimer into the dimeric antiparallel hairpin structure (AP-hairpin). We applied Atomic Force Microscopy (AFM) techniques to follow peptide-peptide interactions at the single molecule level, to visualize the morphology of aggregates formed by both constructs, thioflavin T (ThT) fluorescence to follow the aggregation kinetics, and circular dichroism (CD) spectroscopy to characterize the secondary structure of the constructs. The ThT fluorescence data showed that the AP-hairpin aggregation kinetics is insensitive to the external environment such as ionic strength and pH contrary to the monomers the kinetics of which depends dramatically on the ionic strength and pH. The AFM topographic imaging revealed that AP-hairpins primarily assemble into globular aggregates, whereas linear fibrils are primary assemblies of the monomers suggesting that both constructs follow different aggregation pathways during the self-assembly. These morphological differences are in line with the AFM force spectroscopy experiments and CD spectroscopy measurements, suggesting that the AP-hairpin is structurally rigid regardless of changes of environmental factors.
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Portillo
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA.
authors.forename: Mohtadin
authors.initials: M
authors.lastname: Hashemi
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA.
authors.forename: Yuliang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Molecular Medicine, USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC07, Tampa, FL 33647, USA.
authors.forename: Leonid
authors.initials: L
authors.lastname: Breydo
authors.affiliation: Department of Molecular Medicine, USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC07, Tampa, FL 33647, USA; Department of Biological Science, Faculty of Science, King Abdulaziz University, PO Box 80203, Jeddah 21589, Saudi Arabia; Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Uversky
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA. Electronic address: ylyubchenko@unmc.edu.
authors.forename: Yuri L
authors.initials: YL
authors.lastname: Lyubchenko
mesh_terms: D001665:Binding Sites; D003460:Crystallization / Q000379:methods; D019281:Dimerization; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000648:ultrastructure*; D011485:Protein Binding; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D010454:Peptide Termination Factors; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins
keywords: AFM; Amyloids; Force spectroscopy; Nanoimaging; Neurodegenerative diseases; Protein aggregation