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

A bicyclic undecapeptide of sequence cyclo-(Ala(1)-Pro(2)-Asp(3)-Glu(4)-Lys (5)-Ala(6)-Pro(7)-Asp(8)-Asp(9)-Glu(10))-cyclo-(10 gamma --> 5 epsilon)-Gly (11), deigned to mimic the calcium coordination site I of Clamodulin, has b een synthesized and its conformation and calcium binding properties have be en investigated by means of CD and nmr spectroscopy. The nmr analysis of th e 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 co nfiguration around both Proline residues. The nmr results in both solvents suggest for the molecule a rectangular shape constituted by two antiparalle l beta-strands connected by two beta-turns. Interproton distances, evaluate d by NOE contacts, have been used to obtain feasible models by means of Res trained Molecular Dynamic (RMD). The average models from RMD calculations, for both solvents, exhibit good analogies with Calmodulin site 1. The model system, when compares with the reference system (Asp(20)-Glu(31) segment i n CaM), shows similar dimensions and an effective superimposition of the re ference sequence segments Ala(1)-Glu(4) and Thr(28)-Glu(31). The remaining segments of the model peptide exhibit a bending that is intermediate betwee n that of the free and Ca2+-coordinated site I CD spectra, recorded in TFE solutions, point to a 1:1 stoichimetry for the Ca2+-peptide complex, with a n association constant of at least 1 x 10(5) M-1. (C) 2000 John Wiley & Son s, Inc.