Bicyclic peptides as models of calcium binding sites: Synthesis and conformation of a homodetic undecapeptide

A bicyclic undecapeptide of sequence cyclo-(Ala1-Pro2-Asp3-Glu4-Lys5-Ala6-Pro7- Asp8-Ser9-Glu10)-cyclo-(10γ→5ε)-Gly11, designed to mimic the calcium coordination site 1 of Calmodulin, has been synthesized and its conformation and calcium binding properties have been investigated by means of CD and nmr spectroscopy. The nmr analysis of the free peptide, carried out in DMSO and in TFE/H2O at different pH values, shows the presence in solution of one stable conformer, exhibiting trans configuration around both Proline residues. The nmr results in both solvents suggest for the molecule a rectangular shape constituted by two antiparallel β-strands connected by two β-turns. Interproton distances, evaluated by NOE contacts, have been used to obtain feasible models by means of Restrained Molecular Dynamic (RMD). The average models from RMD calculations, for both solvents, exhibit good analogies with Calmodulin site 1. The model system, when compared with the reference system (Asp20-Glu31 segment in CaM), shows similar dimensions and an effective superimposition of the respective sequence segments Ala1-Glu4 and Thr28-Glu31. The remaining segments of the model peptide exhibit a bending that is intermediate between that of the free and Ca2+-coordinated site 1. CD spectra, recorded in TFE solutions, point to a 1:1 stoichiometry for the Ca2PLU-peptide complex, with an association constant of at least 1×105 M-1.