Polyproline II helix is a key structural motif of the elastic PEVK segmentof titin

Titin is a family of giant elastic proteins that constitute an elastic sarc omere matrix in striated muscle. In the I-band region of the sarcomere, whe re titin extends and develops passive force upon stretch, titin is composed of tandem repeats of similar to 100 residue immunoglobin domains and simil ar to 28-residue PEVK modules. We have performed 2D NMR and circular dichro ism (CD) studies of the conformations of one representative 28-mer PEVK mod ule from human fetal titin (PEPPKEVVPEKKAPVAPPKKPEVPPVKV). NMR data of synt hetic peptides of this module as well as three constituent peptides of 9 to 12 residues in aqueous solutions reveal distinguishing features for left-h anded three-residue per turn PPII helices: the lack of NOE NN(i, i+1), very large NOE alphaN(i, i+1)/NN(i, i+1), no medium range NOE alphaN(i, i+2), a nd dihedral angles phi and psi values of -78 and 146, respectively. Structu ral determinations indicate the presence of three short stretches of PPII h elices of 4, 5, and 6 residues that are interposed with an unordered, and p resumably flexible, spacer region to give one "polyproline II helix-coil" o r "PhC" motif for roughly every 10 residues. These peptides also display th e characteristic PPII CD spectra: positive peak or negative shoulder band a t 223 nm, negative CD band near 200 nm, and biphasic thermal titration curv es that reflect varied stability of these PPII helices. We propose that thi s PhC motif is a fundamental feature and that the number, length, stability , and distribution of PPII is important in the understanding of the elastic ity and protein interactions of the PEVK region of titin.