RESUMO
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.
Assuntos
Comunicação Celular , Tecido Elástico/metabolismo , Tropoelastina/química , Tropoelastina/metabolismo , Adesão Celular , Células Cultivadas , Tecido Elástico/química , Tecido Elástico/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Hidrogéis , Microscopia Confocal , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Tamanho da Partícula , Estrutura Terciária de Proteína , Proteólise , Soluções , Relação Estrutura-Atividade , Temperatura , Tropoelastina/ultraestruturaRESUMO
Tropoelastin is the monomer building block of the biopolymer elastin, which is responsible for elasticity in arteries, lung and skin. Previous studies have shown that, in contrast to predictions made based on primary sequence, tropoelastin has little regular secondary structure in aqueous solution and displays considerable flexibility. This investigation defines the level of residual structure present in tropoelastin and uses the naturally-occurring structure-inducing osmolyte trimethylamine N-oxide to examine the potential for regular structure in tropoelastin. Tropoelastin is defined as a thermodynamically unfolded premolten globule, which can account for its ability to elastically deform.