In the last years polyproline II (PPII) structure has been demonstrated to be essential to biological activities such as signal transduction, transcription, cell motility, and immune response. The polyproline left-handed helical structure was nearly unknown until now and often confused with unordered, disordered, irregular, unstructured, extended, or random coil conformations because it is neither alpha-helical nor beta-turn nor beta-sheet, i.e., a classical structure. In spite of the regularity of the PPII structure and, more precisely, its well-defined dihedral angle values, a typical feature of PPII structure is the absence of any intramolecular hydrogen bonds that renders the PPII structure indistinguishable from an irregular backbone structure by (1)H-NMR spectroscopy. The only way to unambiguously reveal PPII structure in solution is to use spectroscopies based on optical activity, such as circular dichroism (CD), vibrational circular dichroism (VCD), and Raman optical activity (ROA). Herein we focus on the identification of PPII structure by CD, widely considered to be the most reliable methodology. Then we report on VCD and ROA spectroscopies as tools in the identification of PPII structure. A third section is dedicated to the analysis of the stabilization of PPII conformation in aqueous solution. Finally, the significance of PPII in self-assembly processes, in elasticity of elastomeric proteins, and in proteins-(peptides) proteins molecular recognition processes are considered.

Polypeptide sequences encoding the single exons of human tropoelastin were synthesized and their conformations were studied in different solvents and at different temperatures by CD and (1)H NMR. The results demonstrated the presence of labile conformations such as poly-proline II helix (PPII) and beta-turns whose stability is strongly dependent on the microenvironment. Stable, periodic structures, such as alpha-helices, are only present in the poly-alanine cross-linking domains. These findings give a strong experimental basis to the understanding of the molecular mechanism of elasticity of elastin. In particular, they strongly support the description of the native relaxed state of the protein in terms of trans-conformational equilibria between extended and folded structures as previously proposed [Debelle, L., and Tamburro, A. M. (1999) Int. J. Biochem. Cell. Biol. 31, 261-272].

Polyalanine cross-linking domains encoded by exons 6, 15, 17, 19, 21, 23, 25, 27, 29, 31 of human tropoelastin were synthesized, and their conformations were studied in different solutions and at different temperatures by CD and (1)H NMR. The results demonstrated the presence of poly-proline II helix (PPII) in aqueous solvent and of alpha-helical conformation in TFE. The (1)H NMR results allowed the precise localization of the helices along the peptide sequence. These data were further refined by prediction algorithms in order to take into account the reduced helix stability at the end of the peptides. Furthermore, the influence of flanking residues was checked by synthesizing and by determining the structure of a peptide spanning exon 31 coded domain and the first five residues of the following exon 32 coded domain. These studies, together with those previously published [Tamburro, A. M., Bochicchio, B., and Pepe, A. (2003) Biochemistry 42, 13147-62], are used to propose a coherent recomposition of the elastin pieces (domains) in order to give an acceptable solution to the elastin structure-function problem.

Protein-inspired biomaterials have gained great interest as an alternative to synthetic polymers, in particular, for their potential use as biomedical devices. The potential inspiring models are mainly proteins able to confer mechanical properties to tissues and organs, such as elasticity (elastin, resilin, spider silk) and strength (collagen, silk). The proper combination of repetitive sequences, each of them derived from different proteins, represents a useful tool for obtaining biomaterials with tailored mechanical properties and biological functions. In this report we describe the design, the production, and the preliminary characterization of a chimeric polypeptide, based on sequences derived from the highly resilient proteins resilin and elastin and from collagen-like sequences. The results show that the obtained chimeric recombinant material exhibits promising self-assembling properties. Young's modulus of the fibers was determined by AFM image analysis and lies in the range of 0.1-3 MPa in agreement with the expectations for elastin-like and resilin-like materials.

Elastomeric proteins are widespread in the animal kingdom, and their main function is to confer elasticity and resilience to organs and tissues. Besides common functional properties, elastomeric proteins share a common sequence design. They are usually constituted by repetitive sequences with a high content of glycine residues. From a conformational point of view, all the elastomeric proteins since now analyzed show a dynamic equilibria between folded (mainly beta-turns) and extended (polyproline II and beta-strands) conformations that could be at the origin of the high entropy of the relaxed state. As a matter of fact, elastin, lamprin, abductin, as well as the PEVK domain of titin share the same conformational ensemble, thus pointing to a common molecular mechanism as the origin of elasticity. CD spectroscopy represents the proper spectroscopic technique to be used overall because of its particular sensitivity to the presence of PPII structure. Its use in the molecular studies of elastin, abductin, and lamprin as well as the recently analyzed protein resilin will be presented.