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

Abstract

Despite its growing importance in biology and in biomaterials development, liquid-liquid phase separation of proteins remains poorly understood. In particular, the molecular mechanisms underlying simple coacervation of proteins, such as the extracellular matrix protein elastin, have not been reported. Coacervation of the elastin monomer, tropoelastin, in response to heat and salt is a critical step in the assembly of elastic fibers in vivo, preceding chemical crosslinking. Elastin-like polypeptides (ELPs) derived from the tropoelastin sequence have been shown to undergo a similar phase separation, allowing formation of biomaterials that closely mimic the material properties of native elastin. We have used NMR spectroscopy to obtain site-specific structure and dynamics of a self-assembling elastin-like polypeptide along its entire self-assembly pathway, from monomer through coacervation and into a cross-linked elastic material. Our data reveal that elastin-like hydrophobic domains are composed of transient β-turns in a highly dynamic and disordered chain, and that this disorder is retained both after phase separation and in elastic materials. Cross-linking domains are also highly disordered in monomeric and coacervated ELP 3 and form stable helices only after chemical cross-linking. Detailed structural analysis combined with dynamic measurements from NMR relaxation and diffusion data provides direct evidence for an entropy-driven mechanism of simple coacervation of a protein in which transient and nonspecific intermolecular hydrophobic contacts are formed by disordered chains, whereas bulk water and salt are excluded.phase separation | elastin | NMR | protein structure | dynamics T he liquid-liquid phase separation (LLPS) of molecules has long been exploited to concentrate and encapsulate molecules for drug delivery and food preparation (1, 2). In biology, there is increasing awareness that many proteins exhibit this type of phase behavior, allowing transient microenvironments to be quickly assembled and disassembled in response to changing solution conditions, or the availability of binding partners (3, 4). Protein phase separation occurs intracellularly to generate various membraneless organelles, such as ribonucleoprotein (RNP) bodies involved in nucleic acid processing, transport, and storage (5). A similar phenomenon is observed in the extracellular matrix as a critical step in the synthesis of elastic fibers, which provide extensibility, recoil, and resilience to tissues (6, 7). In the latter system, LLPS of monomeric elastin results in hydrated protein-rich coacervate droplets that are deposited and crosslinked to form an elastic matrix (7,8). In addition to their fundamental importance to biology, the dynamic reversibility of LLPS makes phase-separated states of proteins an attractive platform for development of responsive biomaterials with broad application, for instance as scaffolds for tissue engineering or as carriers in drug delivery systems (9-11).Despite the keen interest in proteins that undergo s...

A comprehensive map of human elastin cross‐linking during elastogenesis

“…The observed complexity of the cross-linking pattern in mature elastin suggests that the backbone of TE must retain some mobility in the coacervation process during elastogenesis as a result of a high hydration degree [64]. Additionally, recent studies highlighted the role of high entropy and transient b-turn formation in random coil regions with repetitive amino acid sequences on the self-assembly of intrinsically disordered proteins [65,66]. Considering this together with our results, it is reasonable to assume that the coacervation of TE is characterized by a randomized interpenetration of the polypeptide chains leading to a highly unordered network.…”

Pressure‐Induced Dissolution and Reentrant Formation of Condensed, Liquid–Liquid Phase‐Separated Elastomeric α‐Elastin

“…Depictedare hydrophobic (nonpolar) groups (goldenc ircles) andwater molecules for illustrative purposes (blue circles).E ach elastin molecule is schematicallyr epresented by golden circles connected by at hick blackcurve. [13] It is likely that large numbers of transient voids,t hat is, water-free cavities, are harbored by the dynamic chain configurations in this low-p condensed liquid state, which we refer to as the first condensed phase (Figure 5a). [17d, 20] White areasa re voids.…”

“…This is also in agreement with recentN MR relaxation data, revealing that the entropy-driven mechanism of coacervationentails formation of hydrophobic domains that are composed of transient b-turnsi nahighly dynamic and disordered chain, and that this disorder is retainedi nt he phase separation region. [13] It is likely that large numbers of transient voids,t hat is, water-free cavities, are harbored by the dynamic chain configurations in this low-p condensed liquid state, which we refer to as the first condensed phase (Figure 5a). The situation here should be analogoust ot he presence of significant void volumes in folded proteins [2f] and amyloids [2b, 14] that form stable, compactp olypeptide structures under ambient pressure, althoughv oids in the latter ordered structures are essentially static, whereas those in the condensed liquid phase of elastin are envisioned to be dynamic.…”

Site‐Specific Isotopic Labeling (SSIL): Access to High‐Resolution Structural and Dynamic Information in Low‐Complexity Proteins

“…[47] Unfortunately,t he role of protein structure in triggering or modulating this property is less clear.T hese structurali nvestigations, too, are impaired by the congestion of the NMR spectra of LLPS-inducing LCRs that worsens within droplets. [48][49][50] Future challenges of SSIL To addresst he structuralq uestions described in the previous sections, several challenges will have to be addressed. In this section we will describe those that we think are the most relevant.…”

“…Unfortunately, the role of protein structure in triggering or modulating this property is less clear. These structural investigations, too, are impaired by the congestion of the NMR spectra of LLPS‐inducing LCRs that worsens within droplets …”