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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, 2010Engineering of Amyloid-β-Binding Affibody Molecules forImproved Chemical Synthesis and Higher Binding AffinityJoel Lindgren 1 , Lars Abrahmsén 2 , Sebastian Wärmländer 3 , andAmelie Eriksson Karlström 11 Royal Institute of Technology (KTH), School of Biotechnology, Division of MolecularBiotechnology, AlbaNova University Centre, SE – 106 91, Stockholm, Sweden; 2 AffibodyAB, Lindhagensgatan 133, SE – 112 51, Stockholm, Sweden; 3 Department of Biochemistryand Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University,SE – 106 91, Stockholm, SwedenIntroductionAffibody molecules, based on the engineered domain Z, are a class of affinity proteins withseveral advantageous properties, such as small size (58 aa) and rapid and reversible folding.An Affibody molecule (Z Aβ3 ) previously selected by phage display has been shown tobind Amyloid-β (Aβ) peptides with nanomolar affinity and to inhibit Aβ peptideaggregation [1,2]. The oligomerization and aggregation of Aβ peptides are generallyconsidered to be one of the most important contributors to the onset and progression ofAlzheimer’s disease (AD). Proteins binding Aβ peptides with high affinity and selectivityare therefore important tools to better understand the aggregation process and couldpossibly be used for diagnosis and treatment of AD.Structure analysis of the Aβ peptide binding Affibody molecule showed that Z Aβ3binds as a disulfide-linked homodimer, and that the original three-helix bundle structure ofthe Z domain is lost in the Z Aβ3 Affibody molecule (Figure 1). Instead, the first α-helix ofthe parental protein is found unstructured in the free form of Z Aβ3 , and upon binding to theAβ peptide, residues 15-19 adopt a β-strand conformation [3]. We here describe the design,synthesis and evaluation of six N-terminally truncated variants, together with a full-lengthversion of the Z Aβ3 Affibody molecule [4].Results and DiscussionSix truncated variants of the Z Aβ3 Affibody molecule (Figure 1), together with a full lengthvariant, were successfully synthesized using standard Fmoc SPPS. All variants were foundto be soluble during the purification,workup and analysis of the molecules.The synthetic yields of Z Aβ3 (18-58),Z Aβ3 (15-58) and Z Aβ3 (12-58) were35%, 30% and 29%, respectively,while the yield of the full-lengthprotein, Z Aβ3 (1-58), was only 8% usingthe same reaction conditions.The dissociation constant (K D ) ofthe full-length control Affibodymolecule Z Aβ3 (1-58) was determined toto 9.5 nM. No binding to the Aβ(1-40)peptide could be detected for theshortest variant, Z Aβ3 (18-58). On theFig. 1. The amino acid sequences of the Aβ(1-40)peptide (A) and the ZAβ3(1-58) Affibody molecule(B). The arrows indicate the sites of truncationgiving rise to the seven different variants producedby SPPS. (C) show the previously solved solutionstructure of the Aβ/ ZAβ3 complex. [3] Blockarrows indicate α-strands, while the cylindersindicate α-helical structure, as determined in thestructure analysis of the complex done by Hoyeret al. [3] Figure adopted from [4].other hand, both Z Aβ3 (12-58) andZ Aβ3 (15-58) showed high bindingaffinities with K D values ofrespectively 0.69 nM and 0.48 nM. Themain contributor to the higher affinitiesof both Z Aβ3 (12-58) and Z Aβ3 (15-58)are faster on-rates for the truncatedvariants compared to the full-lengthmolecule Z Aβ3 (1-58) (Table 1).CD-melting profiles were used toinvestigate the effect on the stability ofthe Affibody dimers of addition of178