Dissection of Human Tropoelastin: Exon-By-Exon Chemical Synthesis and Related Conformational Studies

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Abstract
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 ‚-turns whose stability is strongly dependent on the microenvironment. Stable, periodic structures, such as R-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). Resilience and elastic recoil are properties conferred on all vertebrate elastic tissues by elastic fibers ( 1, 2). These complex extracellular matrix biopolymers are composed of two distinct morphological entities. The amorphous compo- nent is made up of insoluble elastin, a highly cross-linked and hydrophobic protein assembled from a soluble precursor protein called tropoelastin. The microfibrillar component of elastic fibers is composed of several glycoproteins, of which the best known are the fibrillins (fibrillin-1 and fibrillin-2) (3, 4). Elastic microfibrils are thought to provide a three- dimensional scaffold for the assembly of elastin during the formation of elastic fibers, while it is insoluble elastin that confers to elastic fibers the property of elastic recoil. The elastic properties of elastin have been explained in terms of entropic components, with questions still remaining about whether the basic mechanism is compatible with the classical theory of rubber elasticity (5). A better understanding of structure-function relationships in terms of the protein's elastic properties remains an important goal in elastin biology. Not only will this provide mechanistic insight into how elastin functions, but may suggest a general mechanism for other elastomeric proteins such as spider silk elastic proteins, glutenin, abductin, etc. ( 6). Furthermore, a complete elucidation of elastin's elasticity is essential for the design of proper biopolymers and biomaterials based on elastomeric proteins (7). The unusual physical properties of elastin have precluded a thorough analysis of the protein's three-dimensional structure at atomic resolution. Mature, cross-linked elastin does not crystallize (as expected for rubber-like material), thereby preventing structural analysis by X-ray diffraction. Similarly, its extreme insolubility in common solvents precludes the use of classical spectroscopic techniques. For these reasons, past studies were confined to the use of soluble derivatives, such as R-elastin (8) and k-elastin (9), and to short synthetic peptides corresponding to repeating sequences