Structural Determinants of Cross-linking and Hydrophobic Domains for Self-Assembly of Elastin-like Polypeptides

Abstract

Elastin is a major structural protein found in large blood vessels, lung, ligaments, and skin, imparting the physical properties of extensibility and elastic recoil to these tissues. To achieve the required structural durability of the elastic matrix, the elastin monomer, tropoelastin, undergoes ordered assembly into a covalently cross-linked, fibrillar polymeric structure. Human tropoelastin consists of 34 exons coding for alternating hydrophobic and cross-linking domains. Using a series of well-defined recombinant polypeptides based on human elastin sequences mimicking native elastin, we have previously investigated the role of sequence and context of hydrophobic domains in elastin self-assembly. Here, we demonstrate that the structure of both cross-linking and hydrophobic domains have significant effects on the assembly of these polypeptides. Removing a putative flexible hinge region in the center of a cross-linking domain substantially increased the alpha-helical content and strongly promoted their self-aggregation. However, while trifluoroethanol (TFE) promoted and urea inhibited self-assembly of these polypeptides, these effects were not predominantly due to altered alpha-helicity of the polypeptides. Our results suggest that, while increased alpha helicity also favors this process, the major effect of TFE to promote organized self-assembly of elastin-like polypeptides is likely related to direct effects of this cosolvent on hydrophobic domains. Such simple elastin polypeptide models can provide an important tool for understanding the relationships between sequence, structure, and polymeric assembly of elastin.

Direct observation of structure and dynamics during phase separation of an elastomeric protein

“…Far UV circular dichroism (CD) spectroscopy of monomeric ELP 3 in aqueous solution gave results consistent with previous studies of tropoelastin and elastin-like polypeptides (28,30,31) (Fig. 1A).…”

“…These data are consistent with the presence of multiple nonspecific intermolecular hydrophobic contacts within the coacervate. The observed interactions between valines and alanines suggest that, whereas the HDs are required for LLPS of ELPs, CLD sequence composition may influence coacervation, supporting previous studies of CLD mutants (28,31).…”

“…We, and others, have shown that elastin-like polypeptides (ELPs) derived from the native sequence of elastin act as suitable proxies to study phase separation and elasticity (18,28,31,39). In the present study, we highlight the design and characterization of an elastin mimetic that undergoes phase separation and can be cross-linked to create resilient biomaterials whose assembly can be monitored by NMR to obtain site-specific resolution of structure and dynamics of both the phase-separated and cross-linked materials.…”

“…The HDs are required for coacervation and elasticity and are disordered domains composed of pseudorepetitive sequence motifs enriched in glycine, proline, and hydrophobic amino acids (20)(21)(22)(23)(24)(25)(26). In contrast, the CLDs provide structural integrity to polymeric elastin through covalent cross-linking of lysine residues (27,28). Current knowledge of elastin structure comes primarily from low-resolution spectroscopic and solid-state NMR studies (29)(30)(31)(32)(33)(34)(35),…”

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

“…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).…”