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Table 1. Thermodynamic and kinetic data for Z Aβ3 (1-58), Z Aβ3 (12-58), Z Aβ3 (15-58) andZ Aβ3 (18-58) binding to the Aβ(1-40) peptide, obtained from CD and SPR measurements.Table adopted from [4]AffibodyTm freeAffibody(°C) bTm Affibodyin complexwith Aβpeptide(°C) bΔTm(°C)k a k d K D(M -1 s -1 ) a (s -1 ) a (nM) aZAβ3(1-58) 50.5 58.1 7.6 4.1 x 10 4 3.9 x 10 -4 9.5 0.8ZAβ3(12-58) 46.2 62.6 16.4 4.5 x 10 5 3.1 x 10 -4 0.7 0.5ZAβ3(15-58) 49.1 63.8 14.7 6.3 x 10 5 3.0 x 10 -4 0.5 1ZAβ3(18-58) 37.9 40.7 2.8 n/a n/a n/a n/aa Determined by SPR measurementsb Determined by CD spectroscopy melting profiles at 220 nmAβ(1-40) peptide. An increased Tm indicates binding and stabilization of the complex. TheT m of Z Aβ3 (12-58) and Z Aβ3 (15-58) are both higher than of the full length variant inpresence of Aβ(1-40), indicating a stronger binding to the Aβ(1-40) peptide (Table 1).The four Affibody variants displayed different transformations of their secondarystructure upon titration with Aβ(1-40). The titration of Aβ(1-40) to Z Aβ3 (18-58) showed noindication of change in the secondary structure of Z Aβ3 (18-58). For Z Aβ3 (1-58), the CDsignal decreases rather uniformly upon addition of Aβ(1-40), indicating loss of helicalstructure. For Z Aβ3 (12-58), the CD signal indicates both a loss of helical content andincreased β-strand structure in the Affibody/Aβ complex upon addition of Aβ(1-40).Finally, for Z Aβ3 (15-58), the CD signal only indicates an increased β-strand content in thesystem when Aβ(1-40) is added. The CD signal of the free Aβ(1-40) peptide was found tobe negligible compared to the signal of the Affibody molecules. This reflects theunstructured nature of free Aβ(1-40), which is characterized by a weak CD signal.However, Aβ(1-40) displays a secondary structure when bound to an Affibody moleculedimer.To conclude, removal of the first 11 and 14 amino acids of Z Aβ3 yields truncatedAffibody molecules Z Aβ3 (12-58) and Z Aβ3 (15-58), respectively, with higher binding affinityto the Aβ (1-40) peptide than the full-length Z Aβ3 (1-58), where the higher on-rate for theN-terminally truncated Affibody variants is the main reason for the higher affinity. This isprobably related to the free full-length Affibody dimer having a partially helicalN-terminus, which must unfold prior to binding the Aβ peptide. Correspondingly to Z Aβ3 ,the truncated versions lock the Aβ peptide in a biologically relevant β-hairpinconformation. The shorter Affibody molecules can be produced in significantly higheryields than the full-length molecule by standard SPPS. This higher yield is an importantadvantage, since chemical production expands the possibilities of making differentconjugates and heterodimeric molecules for further studies of the Affibody molecule/Aβpeptide complex.AcknowledgmentsThis study was supported by grants from the Swedish Natural Science Research Council, the Knut &Alice Wallenberg Foundation, and the SAMBIO program from VINNOVA.References1. Gronwall, C., et al. J. Biotechnol. 128, 162-83 (2007).2. Luheshi, L.M., et al. PLoS. Biol. 8, e1000334 (2010).3. Hoyer, W., et al. Proc Natl. Acad. Sci. U.S.A. 105, 5099-104 (2008).4. Lindgren, J., et al. Protein Sci. In press.χ2179