Kinetics and Morphology of Self-Assembly of an Elastin-like Polypeptide Based on the Alternating Domain Arrangement of Human Tropoelastin

Abstract

Elastin is the polymeric extracellular matrix protein responsible for the properties of extensibility and elastic recoil in tissues such as arterial blood vessels, lung parenchyma, and skin. Both tropoelastin (TE), the full-length monomeric form of elastin, and elastin-like polypeptides (ELPs), based on sequences and domain arrangements of TE, have the intrinsic ability to undergo organized self-assembly into network structures through a process of temperature-induced phase separation or coacervation. It has been suggested that this property plays a role in in vivo formation of the extracellular elastic matrix. In general, the temperature at which phase separation takes place has been taken as the measure of propensity for self-assembly. However, this phase separation is only the first step in a more complex, multistep process of network formation. We have previously shown that analysis of spectrophotometric data allows extraction of kinetic parameters describing both early (coacervation) and later (maturation) steps of the self-assembly process. Here, using a well-characterized ELP containing three hydrophobic domains flanking two cross-linking domains, we describe the effects of temperature, polypeptide concentration, and solution conditions on the kinetics of self-assembly, providing insights into possible mechanisms for the spontaneous organization of such ELPs into extended networks.

Proline Periodicity Modulates the Self-assembly Properties of Elastin-like Polypeptides

“…Although both of these polypeptides exhibited the presence of ␤-structure as dried monomers as well as in the aggregated state, given the disruptive nature of prolines against ␤-sheet formation, it is probable that domain P6N permits a more extended ␤-structure motif than domain P12. Moreover, the introduction of greater spacing between proline residues correlated with the progressive retardation of droplet growth, consistent with more rapid loss of surface fluidity and stabilization of droplet surfaces, preventing coalescence (30,34). Growing evidence shows that water-hydrophobic interfaces promote the self-organization of amphipathic sequences into ␤-sheet structures (53)(54)(55).…”

“…Droplets typically grow (mature) by coalescence before attaining a stable final diameter. The stabilized droplet size is highly dependent on the protein sequence and concentration and on the solvent environment, including the presence of other extracellular matrix proteins (30,34). In this study, the effect of proline spacing on the growth and aggregation characteristics of coacervate droplets was monitored in real time by light microscopy.…”

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 Temperature Correlates with Hydrophobicity and ␤-Sheet Propensity-In general, the coacervation temperature of elastin-like polypeptides has been shown to decrease with increased concentration and molecular weight, as well as with increased solution ionic strength (19,45,57). This phase separation can also be promoted by the addition of relatively low concentrations of TFE to the solution (17).…”

Bioinspired Strategies for Hard Tissue Regeneration

“…This self-assembly property of full-length tropoelastin can also be mimicked by smaller polypeptides. Elastin-like polypeptides (ELPs) have the ability to undergo organized self-assembly into network structures through a process of temperature-induced phase separation or coacervation (20) . Elastin-like polypeptides are derived from a repeating motif within a hydrophobic domain of mammalian tropoelastin: the most common motif has the sequence (VPGXG) m , where X can be any amino acid other than proline, and m is the number of repeats (1).…”