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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010H β2ORH β3NHCD and Fluorescence Screening of α-Synuclein-PeptideInteractionsAnna Marchiani 1 , Giada Massalongo 1 , Stefano Mammi 1 , IsabellaTessari 2 , Luigi Bubacco 2 , Andrea Calderan 1 , and Paolo Ruzza 11 Institute of Biomolecular Chemistry of CNR, Padova Unit, and Department of ChemicalSciences, University of Padova, Padova, 35131, Italy; 2 Department of Biology,University of Padova, Padova, 35131, ItalyIntroductionα-Synuclein (AS) is the major component of the intracellular protein-aggregates found inthe dopaminergic neurons of Parkinson's disease patients.A critical step in the aggregation of AS is the production of oligomers, which are morecytotoxic than the amyloid-like fibrils. Aggregation inhibitors are expected to reduce AScytotoxicity by preventing oligomer formation; on the other hand, an aggregationaccelerator has recently been reported to reduce AS cytotoxicity [1], likely by causingoligomer precipitation. Therefore, ligands that modulate amyloid aggregation may have atherapeutic potential. For this purpose, we synthesized two peptides [2,3], named BB1 andBB2, respectively, and their all-D amino acid analogues as potential amyloid aggregationmodulating ligands. In addition, a rotamer-scan of the Phe4 residue into the BB1 peptidewas performed with the aim to evaluate the influence of the topography of this residue inthe binding process.Constraint-producing amino acid substitutions are typically used as modifier of thepeptide/residue topography. The Tic residue is characterized by only two allowedside-chain orientations, the g(-) or g(+) conformations (Figure 1). The preferred conformationof the D-Tic residue into peptideHNHR 1L-PheH αRORH β3ONHR 1L-TicRNHgauche (-) trans gauche (+)OH αH β2RCH 3NHRR 1OOL-NMePheFig. 1. Structure of constrained analoguesof Phe and staggered side-chainconformers.H β2NHH αH β3chain is g(-), which corresponds to g(+) foran L-residue in the same position [4].For the L-NMePhe residue, all threeside-chain conformations are possible inprinciple, but mostly the t and g(-)conformations are observed in peptides(Figure 1) [5]. The presence of a methylgroup on the nitrogen constrains the ψ angleof the preceding residue to a large valuetypical of those found in extended orβ-structures.In addition to providing topographyrestrictions, constraint residues can impartother properties to peptides, such as increasedresistance to proteolysis.Results and DiscussionPeptides were synthesized by manual SPPS on Rink-amide resin. HBTU/HOBt activationemployed a three-fold molar excess of Fmoc-amino acids in DMF solution for eachcoupling cycle. Coupling to the secondary amino group of constrained residues wasperformed using HATU as the coupling reagent. Deprotection was performed with 20%piperidine. Cleavage from the resin and deprotection were performed by treatment withTFA-anisole-TIS-H 2 O (95:2.5:2.0:0.5 v/v). Peptides were purified by preparativeRP-HPLC and the molecular weights were checked by ESI-MS.Binding of BB1-peptides to AS was monitored by changes in Trp fluorescenceemission (Figure 2A). Peptides (1.5 µM) in 20 mM phosphate buffer, pH 6.8, at 25 °C weretitrated with small aliquots of AS (65.9 µM) with minimal dilution. Peptide fluorescenceemissions were recorded using an excitation wavelength of 295 nm. Binding constantswere estimated from the titration data using a nonlinear least-squares computer fit to theequation based on 1:1 binding stoichiometry [6].Far-UV CD analysis was used both to determine the binding of BB2-peptides to ASand to evaluate the influence of peptide interactions on AS conformation (Figure 2B).332