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Table 1. Peptide sequences and corresponding K d valuesPeptide Sequence K d (µM)BB1 H-Arg-Lys-Val-Phe-Tyr-Thr-Trp-NH 2 2.43±0.21BB1-D H-arg-lys-val-phe-tyr-thr-trp-NH 2 2.12±0.39BB1-MP H-Arg-Lys-Val-NMePhe-Tyr-Thr-Trp-NH 2 2.94±0.36BB1-DMP H-Arg-Lys-Val-NMephe-Tyr-Thr-Trp-NH 2 3.38±0.52BB1-Tic H-Arg-Lys-Val-Tic-Tyr-Thr-Trp-NH 2 4.68±0.59BB1-DTic H-Arg-Lys-Val-tic-Tyr-Thr-Trp-NH 2 5.59±1.20BB1-Ala H-Arg-Lys-Val-Ala-Tyr-Thr-Trp-NH 2 1.04±0.13BB2 H-Arg-Gly-Ala-Val-Val-Thr-Gly-Arg-NH 2 0.50±0.10 aBB2-D H-arg-Gly-ala-val-val-thr-Gly-arg-NH 2 0.35±0.08 aa Determined by CD measurementsFor this purpose, AS (4 µM) in 20 mM phosphate buffer, pH 6.8, at 25 °C was titrated withsmall aliquots of each peptide (200 µM) with minimal dilution. Each CD spectrum wassubtracted of the solvent and peptide contribution, and corrected for dilution. Bindingconstants (Table 1) were estimated from the titration data using a nonlinear least-squarescomputer fit to the equation based on 1:1 binding stoichiometry [7].Fluorescence (a.u.)300A250200150100500350 400 450Wavelength (nm)0.250.490.740.971.261.531.812.172.52ΔA = A L -A R-5,0x10 -5-1,0x10 -4-1,5x10 -4-2,0x10 -4-2,5x10 -4-3,0x10 -4-3,5x10 -40,0 B0.00.31.52.03.05.0190 200 210 220 230 240 250Wavelength (nm)Fig. 2. (A) Fluorescence titration of BB1-Ala peptide by AS, (B) far-UV CD titration of ASby BB2 peptide. Peptide-AS molar ratios are reported inside each figure.CD spectra showed that AS conformation was strongly influenced by the interaction withdesigned peptides. While the interaction with BB1 and BB2 induced an increase of thenegative band at 198 nm, suggesting a corresponding increase of the unorderedconformation of AS, other peptides caused a decrease of this band indicative of a reductionof the random coil conformation. The absence of an ordered CD spectrum suggests thatthese conformational changes are localized in small portions of AS. Surprisingly, bothBB1-MP and BB1-Tic did not affect the far-UV CD spectrum of AS.References1. Bodner, R.A., et al. Proc. Natl. Acad. Sci. U.S.A. 103, 4246-4251 (2006).2. Kokkoni, N., et al. Biochemistry 45, 9906-9918 (2006).3. Palelogou, K.E., Irvine, G.B., El-Agnaf, O.M.A. Biochem. Soc. Trans. 33, 1106-1110 (2005).4. Valle, G., et al. Int. J. Pept. Protein Res. 40, 222-232 (1992).5. Schiller, P.W., et al. Proc. Natl. Acad. Sci. U.S.A 89, 11871-11875 (1992).6. Fan, T.C., et al. J. Biol. Chem. 283, 25468-25474 (2008).7. Siligardi, G., et al. J. Biol. Chem. 277, 20151-20159 (2002).333