Binding of copper(II) ions to the polyproline II helices of PEVK modules of the giant elastic protein titin as revealed by ESI‐MS, CD, and NMR

Abstract

Titin, a family of giant elastic proteins, constitutes an elastic sarcomere matrix in striated muscle. In the I-band region of the sarcomere, the titin PEVK segment acts as a molecular spring to generate elasticity as well as sites of adhesion with parallel thin filaments. Previously, we reported that PEVK consists of tandem repeats of 28 residue modules and that the "polyproline II-coil" motif is the fundamental conformational motif of the PEVK module. In order to characterize the factors that may affect and alter the PPII-coil conformational motifs, we have initiated a systematic study of the interaction with divalent cations (Cu2+, Ca2+, Zn2+, and Ni2+) and a conformational profile of PEVK peptides (a representative 28-mer peptide PR: PEPPKEVVPEKKAPVAPPKKPEVPPVKV and its subfragments PR1: kvPEPPKEVVPE, PR2: VPEKKAPVAPPK, PR3: KPEVPPVKV). UV-Vis absorption difference spectra and CD spectra showed that Cu2+ bound to PR1 with high affinity (20 microM), while its binding to PR2 and PR3 as well as the binding of other cations to all four peptides were of lower affinity (>100 microM). Conformational studies by CD revealed that Cu2+ binding to PR1 resulted in a polyproline II to turn transition up to a 1:2 PR1/Cu2+ ratio and a coil to turn transition at higher Cu2+ concentration. ESI-MS provided the stoichiometry of PEVK peptide-Cu2+ complexes at both low and high ion strength, confirming the specific high affinity binding of Cu2+ to PR1 and PR. Furthermore, NMR and ESI-MS/MS fragmentation analysis elucidated the binding sites of the PEVK peptide-Cu2+ complexes at (-2)KVPE2, 8VPE10, 13APV15, and 22EVP24. A potential application of Cu2+ binding in peptide sequencing by mass spectrometry was also revealed. We conclude that Cu2+ binds and bends PEVK peptides to a beta-turn-like structure at specific sites. The specific targeting of Cu2+ towards PPII is likely to be of significant value in elucidating the roles of PPII in titin elasticity as well as in interactions of proline-rich proteins.

The 1.49Å Resolution Crystal Structure of PsbQ from Photosystem II of Spinacia oleracea Reveals a PPII Structure in the N-terminal Region

“…This ion has been found to be capable of binding both the ring N of the prolyl residue in a series of substituted polyproline 36 and PPII regions of synthesized polypeptides. 37 Besides, it has been reported 36 that Cu 2C leads to a conformational change of PPII regions to other secondary structure elements (i.e. b-turn-like structure) on increasing concentrations.…”

Structure of giant muscle proteins

“…Cu 2+ binds to specific regions in the PEVK repeat and alters the structure from a polyproline helix to β-turn (Ma and Wang, 2003a). Additionally, changes in environment, such as dielectric constant or temperature, transition the PEVK structure from a polyproline helix to a β turn or to a random coil (Ma et al, 2001; Ma and Wang, 2003a,b). Likewise, protein binding can significantly influence PEVK structure and therefore elasticity (Ma and Wang, 2002; Ma et al, 2006).…”

Facile aqueous synthesis and stabilization of nearly monodispersed gold nanospheres by poly(L‐proline)

“…Proline is the only amino acid that has been used as a ligand in asymmetric catalysis,26 and the binding of copper (II) ions in the PLP II helices in proteins is well documented 27. In addition, oligoprolines are used as ligands in metal complexes in electrochemical reactions, where it has been observed that the redox potential28 of the ferrocenyl group attached to the oligoproline chains depends on the sequence and length of oligoprolines. Finally, a recent study showed that a 50% proline‐containing cell‐penetrating hexapeptide exhibited enhanced translocation across the cytoplasmic membrane as Au‐NP transporting cargo 29…”