Localizing α-Helices in Human Tropoelastin:  Assembly of the Elastin “Puzzle”

Abstract

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.

Conformational Transitions of the Cross-linking Domains of Elastin during Self-assembly

“…Observed helical content, measured by CD or NMR, is consistently lower than would be expected for fully formed helical structures (4,16,17,35). Indeed, fully formed ␣-helices were only observed on the addition of known helixinducing solvents such as trifluoroethanol (TFE) 2 (4,16). Thus, it remains unclear whether interaction of lysine side chains in the cross-linking domains is entirely dependent on alignment of hydrophobic domains or may be the result of stochastic fluctuations in structure that lead to juxtaposition of lysine side chains.…”

“…Observed helical content, measured by CD or NMR, is consistently lower than would be expected for fully formed helical structures (4,16,17,35). Indeed, fully formed ␣-helices were only observed on the addition of known helixinducing solvents such as trifluoroethanol (TFE) 2 (4,16).…”

“…Cross-linking domains can be separated into two specific sequence types, those consisting of lysines located within a proline-rich sequence (KP-type) or the more abundant type that contains polyalanine sequences interspersed with lysines in the form of KAAK or KAAAK (KA-type). KA-type cross-linking domains have been suggested to form an ␣-helical secondary structure that is believed to facilitate formation of crosslinks by bringing lysine residues together on the same face of the helix (4,(15)(16)(17)(18).…”

Elastin, a Novel Extracellular Matrix Protein Adhering to Mycobacterial Antigen 85 Complex

“…The atomic coordinates for the 15 best of 100 NMR conformers of pHTE 27 was generously provided by Antonietta Pepe and Brigida Bochicchio (35). The lowest root mean standard deviation pHTE 27 NMR structure from the set of 15 was used for docking.…”

High resolutionq‐space imaging studies of water in elastin

“…15,16 Polyalanine cross-link domains, encoded by several exons of human tropoelastin, exhibited poly-proline II helix and a-helix conformation in aqueous and trifluoroethanol solutions, respectively. 17 Several NMR dynamic studies have also pointed to elastin being highly mobile with the association of water. [18][19][20] The NMR relaxation times of waters of hydration in elastin have been studied, and it was suggested that two distinct water components exist within elastin fibers; tightly bound waters of hydration in addition to water having more ''bulk-like'' properties reminiscent of free water.…”