The tropoelastin monomer undergoes stages of association by coacervation, deposition onto microfibrils, and cross-linking to form elastic fibers. Tropoelastin consists of an elastic N-terminal coil region and a cell-interactive C-terminal foot region linked together by a highly exposed bridge region. The bridge region is conveniently positioned to modulate elastic fiber assembly through association by coacervation and its proximity to dominant cross-linking domains. Tropoelastin constructs that either modify or remove the entire bridge and downstream regions were assessed for elastogenesis. These constructs focused on a single alanine substitution (R515A) and a truncation (M155n) at the highly conserved arginine 515 site that borders the bridge. Each form displayed less efficient coacervation, impaired hydrogel formation, and decreased dermal fibroblast attachment compared to wild-type tropoelastin. The R515A mutant protein additionally showed reduced elastic fiber formation upon addition to human retinal pigmented epithelium cells and dermal fibroblasts. The small-angle X-ray scattering nanostructure of the R515A mutant protein revealed greater conformational flexibility around the bridge and C-terminal regions. This increased flexibility of the R515A mutant suggests that the tropoelastin R515 residue stabilizes the structure of the bridge region, which is critical for elastic fiber assembly.tropoelastin assembly | protease resistance E lastic fibers confer the elastic and recoil properties required for repetitive and reversible deformation of elastic tissues during normal function (1-3). The assembly of elastic fibers from tropoelastin, the soluble precursor of elastin, is classically defined by stages of tropoelastin synthesis, coacervation, microfibrillar deposition, and cross-linking. Tropoelastin is secreted by elastogenic cells such as smooth muscle cells, endothelial cells, and fibroblasts (1, 2). At the cell surface, the monomers cluster through hydrophobic domain interactions in an aqueous environment (3-6) by the entropically driven process of coacervation (7). These tropoelastin assemblies remain attached through the C terminus to cell-surface integrin αvβ3 and glycosaminoglycans (8-10) until deposition on microfibrillar scaffolds (11), which direct the shape and orientation of elastic fibers (10, 12, 13). Microfibrillar proteins recruit lysyl oxidase (14, 15), which reacts with specific tropoelastin lysine residues to form cross-links (1, 11, 16). These cross-links occur at multiple sites in the molecule (17) and are enriched in domains 19-25 (1, 16). Cross-linking imposes expansional constraints on elastin and renders elastic fibers resilient under repetitive mechanical stretching (1,12,18).The recently solved nanostructure of tropoelastin has allowed the main functional regions of tropoelastin to be placed within a structural context (19). Most of the elasticity of the molecule is conferred by a coiled region (20) that extends from domain 2 to domain 18. A protrusion from the coil region around do...

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