Stages in tropoelastin coalescence during synthetic elastin hydrogel formation

Tu, Yidong; Wise, Steven G.; Weiss, Anthony S.

doi:10.1016/j.micron.2009.11.003

Cited by 53 publications

(57 citation statements)

References 22 publications

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“…ELP and elastin coacervate droplets have been shown to increase in stability if they are held above the coacervation temperature for an extended period (18,48,49). This maturation process has been hypothesized to occur through increased ordering of ELPs at the surface of the droplet, resulting in a less reversible LLPS.…”

Section: Resultsmentioning

confidence: 99%

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

Reichheld

Muiznieks

Keeley

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

212

245

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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...

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Section: Resultsmentioning

confidence: 99%

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

Reichheld

Muiznieks

Keeley

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

212

245

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“…In this respect, it is interesting to note that coacervate droplets of full-length tropoelastin, which includes domain 30, are small and show a significant tendency to cluster (31,60). Moreover, in vitro imaging of hydrogel formation after coacervation of human tropoelastin has described network formation through clustering and partial coalescence of coacervate spheres (61). Thus, formation of organized fibrillar structures from coacervates of tropoelastin, in vivo or in vitro, likely requires modulation of surface properties of coacervate droplets, limiting complete coalescence but allowing some fusion and interdigitation of droplet surfaces.…”

Section: Proline Prevents the Aggregation Of Exposed Hydrophobicmentioning

confidence: 99%

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

Muiznieks

Keeley

2010

Journal of Biological Chemistry

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Elastin is a self-assembling protein of the extracellular matrix that provides tissues with elastic extensibility and recoil. The monomeric precursor, tropoelastin, is highly hydrophobic yet remains substantially disordered and flexible in solution, due in large part to a high combined threshold of proline and glycine residues within hydrophobic sequences. In fact, proline-poor elastin-like sequences are known to form amyloidlike fibrils, rich in ␤-structure, from solution. On this basis, it is clear that hydrophobic elastin sequences are in general optimized to avoid an amyloid fate. However, a small number of hydrophobic domains near the C terminus of tropoelastin are substantially depleted of proline residues. Here we investigated the specific contribution of proline number and spacing to the structure and self-assembly propensities of elastin-like polypeptides. Increasing the spacing between proline residues significantly decreased the ability of polypeptides to reversibly self-associate. Real-time imaging of the assembly process revealed the presence of smaller colloidal droplets that displayed enhanced propensity to cluster into dense networks. Structural characterization showed that these aggregates were enriched in ␤-structure but unable to bind thioflavin-T. These data strongly support a model where proline-poor regions of the elastin monomer provide a unique contribution to assembly and suggest a role for localized ␤-sheet in mediating self-assembly interactions.Elastin is the extracellular matrix protein responsible for the elasticity of vertebrate arterial vessels, connective tissues, lung, and skin. Elastin resilience is afforded by the formation of insoluble fibers, the first step of which involves the selfalignment of elastin monomers, tropoelastin, in a temperature-induced transition known as coacervation, through the association of hydrophobic domains (1, 2). Although highly (Ͼ75%) non-polar in character, tropoelastin remains predominantly monomeric and structurally disordered in solution (3-5) and critically, retains substantial backbone hydration and flexibility even when assembled (6) and cross-linked into mature, polymeric elastic arrays (7-13).Maintenance of structural heterogeneity and dynamics of the elastin backbone is achieved in large part by a high proportional composition of both proline (14%) and glycine (35%) residues within hydrophobic sequences (6, 14). These residues are commonly arranged into repeated motifs based on recurring sequence elements such as PGV and GVA. This includes a seven-fold tandem PGVGVA repeat in domain 24 of the native human elastin sequence (15). Contrary to enhancing coacervation or elastomeric properties, the multiple substitution of glycine residues for prolines within tandem repeat sequences (e.g. PGVGVA to GGVGVA) results in the formation of amyloid-like fibrils, rich in ␤-structure, from solution (6, 14). These structures are conformationally more restricted than native elastomeric sequences as a result of the tighter packing of residues into extended...

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“…As compared to chemically crosslinked polymers, the gelation of the enzyme hydrogels occurs rather slowly, similar as observed for synthetic elastin hydrogels, where the polymerising proteins first form discrete particles that merge to form larger spheres and further coalesce into open linked networks of a porous hydrogel network 31 . To further elucidate their material properties, the enzyme hydrogels were analysed by optical microrheology based on multiple particle tracking (MPT) analysis 32 (Supplementary Figure 4).…”

Section: Resultsmentioning

confidence: 62%

Self-assembling all-enzyme hydrogels for biocatalytic flow processes

Peschke

Gallus

Bitterwolf

et al. 2017

Preprint

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______________________________________________________________We describe the construction of binary self-assembling all-enzyme hydrogels that are comprised entirely of two tetrameric globular enzymes, the stereoselective dehydrogenase LbADH and the cofactor-regenerating glucose 1-dehydrogenase GDH. The enzymes were genetically fused with a SpyTag or SpyCatcher domain, respectively, to generate two complementary homo-tetrameric building blocks that polymerise under physiological conditions into porous hydrogels. The biocatalytic gels were used for the highly stereoselective reduction of a prochiral diketone substrate where they showed the typical behaviour of the coupled kinetics of coenzyme regenerating reactions in the substrate channelling regime. They effectively sequestrate the NADPH cofactor even under continuous flow conditions. Owing to their sticky nature, the gels can be readily mounted in simple microfluidic reactors without the need for supportive membranes. The reactors revealed extraordinary high space-time yields with nearly quantitative conversion (>95%), excellent stereoselectivity (d.r. > 99:1), and total turnover numbers of the expensive cofactor NADP(H) that are amongst the highest values ever reported. ______________________________________________________________

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Stages in tropoelastin coalescence during synthetic elastin hydrogel formation

Cited by 53 publications

References 22 publications

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

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

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

Self-assembling all-enzyme hydrogels for biocatalytic flow processes

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