Tropoelastin Massively Associates during Coacervation To Form Quantized Protein Spheres

Abstract

Tropoelastin, the precursor of elastin, undergoes a rapid monomer to multimer association in an inverse temperature transition. This association culminates in the rapid formation of stable, optically distinct droplets of tropoelastin. Light scattering and microscope measurements reveal that these droplets are 2-6 microm in diameter. Scanning electron microscopy confirms that the droplets are spherical. Three-dimensional confocal image stacks based on the autofluorescence of tropoelastin reveal that droplets are loaded with hydrated tropoelastin. Droplets are viable intermediates in synthetic elastin macroassembly. Dense clusters of aggregated droplets and partially formed fibers develop when droplets are incubated in the presence of a lysyl oxidase. Lysine-reacting chemical and enzyme-assisted cross-linking conditions generate cross-linked beads due to interactions between multiple, surface-exposed lysine epsilon-amino groups. Droplets represent an efficient mechanism for the bolus delivery during elastogenesis of quantized packages of preaccreted tropoelastin.

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

“…We and others have previously shown that colloidal droplets in suspensions held above the coacervation temperature undergo a maturation process involving growth by coalescence and/or clustering (21,29,56,57). We therefore used light microscopy to compare maturation morphologies of the coacervate droplets formed by these three polypeptides.…”

“…Coacervation is an endothermic, entropically driven process (24 -26) through which hydrophobic domain interaction has been suggested to concentrate and align cross-linking domains for polymerization (5,22,27,28). In contrast to other forms of protein aggregation that create highly ordered insoluble structures, such as ␤-strand stacking in amy-loid fiber formation, coacervation forms viscous protein-rich colloidal droplets that can interact and coalesce (29,30). Unlike amyloid fibril formation, coacervation may be reversed in vitro by immediately lowering the temperature of the colloidal suspension (20,30).…”

New insights into elastic fiber assembly

“…This high degree of cross-linking is responsible for the stability and insolubility of the protein. Tropoelastin is capable of undergoing coacervation under physiological conditions in a process thought to facilitate selfassembly (Cox et al, 1974;Volpin et al, 1976;Clarke et al, 2006). Coacervation is the result of specific interactions between the hydrophobic domains induced by an increase in temperature (Toonkool et al, 2001).…”

Contribution of lysine-containing cationic domains to thermally-induced phase transition of elastin-like proteins and their sensitivity to different stimuli

“…The hydrophobic domains are composed of repeats of alternating hydrophobic amino acid sequences such as VPGVG and APGVGV, whereas the AK cross-linking domains consist mainly of Ala and Lys, e.g., AAAAKAAKYGA (3). Under physiological conditions, the hydrophobic domains of tropoelastin undergo a self-assembly process that is referred to as coacervation (4), and the resulting assembly is stabilized by the formation of intermolecular crosslinks between Lys residues that produce a highly insoluble network of elastic fibers (5).…”