CONFORMATIONAL CONSEQUENCES OF I,I- NUCLEATION FOR ALPHA-HELICITY(3 CYSTINE LINKAGES )

Methods to introduce specific secondary structural elements into pepti des and proteins are vital fur the rational design of peptide and non- peptide drug, candidates as well as in the Lie novo design of proteins , Here the incorporation of a disulfide linkage between cysteine resid ues spaced three amino acids apart (i, i+3) as a method to induce heli city is examined. Two dodecamer peptides, A-V-S-E-C-Q-L-C-H-D-K-G-NH2, differing in the chirality of the cysteine at the fifth position (the i position. have been synthesized and conformationally studied both i n the linear and cyclized form. This peptide sequence, derived from th e N-terminal sequence of parathyroid hormone related protein, does not form helices, even as part of the 1-34 fully active domain of the pro tein. The four analogs (two cyclic and two linear) were analyzed both in aqueous solution and in the presence of sodium dodecyl sulfate mice lles. In aqueous solution the linear peptides display no evidence for secondary structure, while the cyclization induces a turn centered abo ut the cysteine residues, In the presence of micelles the linear form of the peptides adopts bent conformations, containing turns, but resul ts from both NMR and CD provide no evidence of helices. The oxidized L ,L-peptide in the micellar solution does not present a well defined co nformation. although the presence of one helical turn is evident. The cyclic D,L analog adopts a helical structure (not an alpha-helix) exte nding from residue 2 to 9, with non-standard phi, psi values caused by the presence of the D-amino acid. These results clearly illustrate th at the ability of D-Cys(i), Cys(i+3) cyclization to initiate helix for mation depends greatly on the solvent used. Therefore, ally drug-desig n principle utilizing this modification for helix nucleation must keep the environment in which the peptide is biologically active in mind. (C) Munksgaard 1997.