The evolutionary background and functional consequences of the rs2071307 polymorphism in human tropoelastin.

Elastin is a major polymeric protein of the extracellular matrix, providing critical properties of extensibility and elastic recoil. The rs2071307 genomic polymorphism, resulting in the substitution of a serine for a glycine residue in a VPG motif in tropoelastin, has an unusually high minor allele frequency in humans. A consequence of such allelic heterozygosity would be the presence of a heterogeneous elastin polymer in up to 50% of the population, a situation which appears to be unique to Homo sapiens. VPG motifs are extremely common in hydrophobic domains of tropoelastins and are the sites of transient ß-turns that are essential for maintaining the conformational flexibility required for its function as an entropic elastomer. Earlier data demonstrated that single amino acid substitutions in tropoelastin can have functional consequences for polymeric elastin, particularly when present in mixed polymers. Here, using NMR and molecular dynamics approaches, we show the rs2071307 polymorphism reduces local propensity for ß-turn formation, with a consequent increase in polypeptide hydration and an expansion of the conformational ensemble manifested as an increased hydrodynamic radius, radius of gyration and asphericity. Furthermore, this substitution affects functional properties of polymeric elastin, particularly in heterogeneous polymers mimicking allelic heterozygosity. We discuss whether such effects, together with the unusually high minor allele frequency of the polymorphism, could imply some some evolutionary advantage for the heterozygous state.

Sequence variants of human tropoelastin affecting assembly, structural characteristics and functional properties of polymeric elastin in health and disease.

Elastin is the polymeric protein responsible for the physiologically important properties of extensibility and elastic recoil of cardiovascular, pulmonary and many other tissues. In spite of significant advances in the understanding how monomeric tropoelastin is assembled into the polymeric elastic matrix, details of this assembly process are still lacking. In particular it is not clear how the various architectures and more subtle elastic properties required by diverse elastic tissues can arise from the protein product of a single gene. While monomeric tropoelastin has the intrinsic ability to self-assemble into fibrillar structures, it is clear that in vivo assembly is guided by interactions with cells and other matrix-associated components. In addition, the multiplicity of reported mRNA isoforms of human tropoelastin, if translated into protein variants, could modulate not only interactions with these matrix-associated components but also self-assembly and functional properties. Critical information identifying such protein isoforms of human tropoelastin is only now emerging from mass spectrometric studies. Increased levels of complexity of the assembly process provide additional opportunities for production of polymeric elastins with aberrant architectures and sub-optimal functional properties that could affect the longer-term structural integrity of elastic matrices. Biophysical techniques, such as SAXS, NMR and molecular dynamics, have provided a means to discern details of the effects of sequence variants, including both alternate splicing isoforms and genetic polymorphisms, on the dynamic flexibility of elastin required for its elastomeric properties. Such approaches promise to provide important new insights into the relationship between sequence, structural characteristics, assembly and functional properties of elastin in both health and disease.

Role of Liquid-Liquid Phase Separation in Assembly of Elastin and Other Extracellular Matrix Proteins.

Liquid-liquid phase separation resulting in formation of colloidal droplets has recently attracted attention as a mechanism for rapid and transient assembly of intracellular macromolecules into functional units. Phase separation also appears to be a widespread and evolutionarily ancient mechanism for organization of proteins of the extracellular matrix into fibrillar, polymeric assemblies. Elastin, which provides the physical properties of extensibility and elastic recoil to large arteries, lungs and other tissues, is the best-characterized extracellular matrix protein whose polymeric assembly is initiated by phase separation. Recent studies have provided an atomistic description of the conformational ensemble of elastin-like proteins, and have begun to uncover how the interplay of local secondary structure, hydrophobicity and conformational disorder govern the structure, assembly and function of elastin. Monomeric elastin is a non-polar, glycine-rich, low-complexity, modular protein that remains predominantly disordered even in the crosslinked polymeric state, consistent with its function as an entropic elastomer. Unlike intracellular phase separation, which is reversible, phase separation of elastin and other matrix proteins proceeds to stabilization and clustering of condensed phase droplets and subsequent molecular organization into fibrillar, supramolecular structures. Short ß-sheets appear to mediate the interaction and organization of these phase-separated droplets and modulate the ultimate material properties of the matrix. Whether phase separation is intracellular or extracellular, reversible or network-forming, understanding the sequence determinants of such varied assembly behaviors and differential fates of the colloidal droplets will provide important insights into aberrant assembly with pathological consequences and elucidate fundamental principles for the rational design of biomimetic materials.

The Personal Genome Project Canada: findings from whole genome sequences of the inaugural 56 participants.

BACKGROUND: The Personal Genome Project Canada is a comprehensive public data resource that integrates whole genome sequencing data and health information. We describe genomic variation identified in the initial recruitment cohort of 56 volunteers. METHODS: Volunteers were screened for eligibility and provided informed consent for open data sharing. Using blood DNA, we performed whole genome sequencing and identified all possible classes of DNA variants. A genetic counsellor explained the implication of the results to each participant. RESULTS: Whole genome sequencing of the first 56 participants identified 207 662 805 sequence variants and 27 494 copy number variations. We analyzed a prioritized disease-associated data set (n = 1606 variants) according to standardized guidelines, and interpreted 19 variants in 14 participants (25%) as having obvious health implications. Six of these variants (e.g., in BRCA1 or mosaic loss of an X chromosome) were pathogenic or likely pathogenic. Seven were risk factors for cancer, cardiovascular or neurobehavioural conditions. Four other variants - associated with cancer, cardiac or neurodegenerative phenotypes - remained of uncertain significance because of discrepancies among databases. We also identified a large structural chromosome aberration and a likely pathogenic mitochondrial variant. There were 172 recessive disease alleles (e.g., 5 individuals carried mutations for cystic fibrosis). Pharmacogenomics analyses revealed another 3.9 potentially relevant genotypes per individual. INTERPRETATION: Our analyses identified a spectrum of genetic variants with potential health impact in 25% of participants. When also considering recessive alleles and variants with potential pharmacologic relevance, all 56 participants had medically relevant findings. Although access is mostly limited to research, whole genome sequencing can provide specific and novel information with the potential of major impact for health care.

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

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 cross-linking. 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 ß-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 ELP3 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.

Biomechanical Design of Elastic Protein Biomaterials: A Balance of Protein Structure and Conformational Disorder.

Elastic biomaterials are found across biology where they fulfill diverse load-bearing and energy storage and dissipation functions. This class of biomaterials comprises elastic proteins that provide materials with combinations of extensibility, stiffness, tensile strength, toughness, and viscoelastic properties. Differences in mechanical properties are due in large part to variations in the ratio of secondary structure and conformational disorder of constituent protein monomers, arising from differences in amino acid sequence. This natural diversity provides rich inspiration for the design of elastic biomaterials. Here, we review the relationship between sequence, structure, disorder, and mechanical properties of elastic proteins from natural materials ranging from highly extensible and soft, to mechanically strong and tough. We describe molecular strategies as well as recombinant efforts to design materials with tailored mechanical properties, with the ultimate aim of rationally engineering biomaterials for advanced biomedical applications.

Single nucleotide polymorphisms and domain/splice variants modulate assembly and elastomeric properties of human elastin. Implications for tissue specificity and durability of elastic tissue.

Polymeric elastin provides the physiologically essential properties of extensibility and elastic recoil to large arteries, heart valves, lungs, skin and other tissues. Although the detailed relationship between sequence, structure and mechanical properties of elastin remains a matter of investigation, data from both the full-length monomer, tropoelastin, and smaller elastin-like polypeptides have demonstrated that variations in protein sequence can affect both polymeric assembly and tensile mechanical properties. Here we model known splice variants of human tropoelastin (hTE), assessing effects on shape, polymeric assembly and mechanical properties. Additionally we investigate effects of known single nucleotide polymorphisms in hTE, some of which have been associated with later-onset loss of structural integrity of elastic tissues and others predicted to affect material properties of elastin matrices on the basis of their location in evolutionarily conserved sites in amniote tropoelastins. Results of these studies show that such sequence variations can significantly alter both the assembly of tropoelastin monomers into a polymeric network and the tensile mechanical properties of that network. Such variations could provide a temporal- or tissue-specific means to customize material properties of elastic tissues to different functional requirements. Conversely, aberrant splicing inappropriate for a tissue or developmental stage or polymorphisms affecting polymeric assembly could compromise the functionality and durability of elastic tissues. To our knowledge, this is the first example of a study that assesses the consequences of known polymorphisms and domain/splice variants in tropoelastin on assembly and detailed elastomeric properties of polymeric elastin.

Phase separation and mechanical properties of an elastomeric biomaterial from spider wrapping silk and elastin block copolymers.

Elastin and silk spidroins are fibrous, structural proteins with elastomeric properties of extension and recoil. While elastin is highly extensible and has excellent recovery of elastic energy, silks are particularly strong and tough. This study describes the biophysical characterization of recombinant polypeptides designed by combining spider wrapping silk and elastin-like sequences as a strategy to rationally increase the strength of elastin-based materials while maintaining extensibility. We demonstrate a thermo-responsive phase separation and spontaneous colloid-like droplet formation from silk-elastin block copolymers, and from a 34 residue disordered region of Argiope trifasciata wrapping silk alone, and measure a comprehensive suite of tensile mechanical properties from cross-linked materials. Silk-elastin materials exhibited significantly increased strength, toughness, and stiffness compared to an elastin-only material, while retaining high failure strains and low energy loss upon recoil. These data demonstrate the mechanical tunability of protein polymer biomaterials through modular, chimeric recombination, and provide structural insights into mechanical design. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 693-703, 2016.

Evolution of the fish heart by sub/neofunctionalization of an elastin gene.

The evolution of phenotypic traits is a key process in diversification of life. However, the mechanisms underlying the emergence of such evolutionary novelties are largely unknown. Here we address the origin of bulbus arteriosus (BA), an organ of evolutionary novelty seen in the teleost heart outflow tract (OFT), which sophisticates their circulatory system. The BA is a unique organ that is composed of smooth muscle while the OFTs in other vertebrates are composed of cardiac muscle. Here we reveal that the teleost-specific extracellular matrix (ECM) gene, elastin b, was generated by the teleost-specific whole-genome duplication and neofunctionalized to contribute to acquisition of the BA by regulating cell fate determination of cardiac precursor cells into smooth muscle. Furthermore, we show that the mechanotransducer yap is involved in this cell fate determination. Our findings reveal a mechanism of generating evolutionary novelty through alteration of cell fate determination by the ECM.

Contribution of domain 30 of tropoelastin to elastic fiber formation and material elasticity.

Elastin is a fibrous structural protein of the extracellular matrix that provides reversible elastic recoil to vertebrate tissues such as arterial vessels, lung, and skin. The elastin monomer, tropoelastin, contains a large proportion of intrinsically disordered and flexible hydrophobic sequences that collectively are responsible for the initial phase separation of monomers during assembly, and are essential for driving elastic recoil. While structural disorder of hydrophobic sequences is controlled by a high proline and glycine residue composition, hydrophobic domain 30 of human tropoelastin is atypically proline-poor, and forms ß-sheet amyloid-like fibrils as an individual peptide. We explored the contribution of confined regions of secondary structure at the location of domain 30 in human tropoelastin to fiber assembly and mechanical properties using a set of mutations designed to inhibit or enhance the propensity of ß-sheet formation at this location. Our data support a dual role for confined ß-sheet secondary structure in domain 30 of tropoelastin in guiding the formation of fibers, and as a determinant of stiffness and viscoelastic properties of cross-linked materials. Together, these results suggest a mechanism for specificity in fiber assembly, and elucidate structure-function relationships for the rational design of elastomeric biomaterials with defined mechanical properties.

Proline-poor hydrophobic domains modulate the assembly and material properties of polymeric elastin.

Elastin is a self-assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline-poor and, as an isolated polypeptide, is susceptible to formation of amyloid-like structures comprised of stacked ß-sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin-like polypeptides designed with different numbers of proline-poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline-poor hydrophobic sequences to self-assembly through characterization of phase separation, and to the tensile properties of cross-linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form ß-structure, both affecting polypeptide chain flexibility and cross-link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence-structure-function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties.

Molecular-level characterization of elastin-like constructs and human aortic elastin.

This study aimed to characterize the structures of two elastin-like constructs, one composed of a cross-linked elastin-like polypeptide and the other one of cross-linked tropoelastin, and native aortic elastin. The structures of the insoluble materials and human aortic elastin were investigated using scanning electron microscopy. Additionally, all samples were digested with enzymes of different specificities, and the resultant peptide mixtures were characterized by ESI mass spectrometry and MALDI mass spectrometry. The MS(2) data was used to sequence linear peptides, and cross-linked species were analyzed with the recently developed software PolyLinX. This enabled the identification of two intramolecularly cross-linked peptides containing allysine aldols in the two constructs. The presence of the tetrafunctional cross-link desmosine was shown for all analyzed materials and its quantification revealed that the cross-linking degree of the two in vitro cross-linked materials was significantly lower than that of native elastin. Molecular dynamics simulations were performed, based on molecular species identified in the samples, to follow the formation of elastin cross-links. The results provide evidence for the significance of the GVGTP hinge region of domain 23 for the formation of elastin cross-links. Overall, this work provides important insight into structural similarities and differences between elastin-like constructs and native elastin. Furthermore, it represents a step toward the elucidation of the complex cross-linking pattern of mature elastin.

Modulated growth, stability and interactions of liquid-like coacervate assemblies of elastin.

Elastin self-assembles from monomers into polymer networks that display elasticity and resilience. The first major step in assembly is a liquid-liquid phase separation known as coacervation. This process represents a continuum of stages from initial phase separation to early growth of droplets by coalescence and later "maturation" leading to fiber formation. Assembly of tropoelastin-rich globules is on pathway for fiber formation in vivo. However, little is known about these intermediates beyond their size distribution. Here we investigate the contribution of sequence and structural motifs from full-length tropoelastin and a set of elastin-like polypeptides to the maturation of coacervate assemblies, observing their growth, stability and interaction behavior, and polypeptide alignment within matured globules. We conclude that maturation is driven by surface properties, leading to stabilization of the interface between the hydrophobic interior and aqueous solvent, potentially through structural motifs, and discuss implications for droplet interactions in fiber formation.

Conformational transitions of the cross-linking domains of elastin during self-assembly.

Elastin is the intrinsically disordered polymeric protein imparting the exceptional properties of extension and elastic recoil to the extracellular matrix of most vertebrates. The monomeric precursor of elastin, tropoelastin, as well as polypeptides containing smaller subsets of the tropoelastin sequence, can self-assemble through a colloidal phase separation process called coacervation. Present understanding suggests that self-assembly is promoted by association of hydrophobic domains contained within the tropoelastin sequence, whereas polymerization is achieved by covalent joining of lysine side chains within distinct alanine-rich, α-helical cross-linking domains. In this study, model elastin polypeptides were used to determine the structure of cross-linking domains during the assembly process and the effect of sequence alterations in these domains on assembly and structure. CD temperature melts indicated that partial α-helical structure in cross-linking domains at lower temperatures was absent at physiological temperature. Solid-state NMR demonstrated that ß-strand structure of the cross-linking domains dominated in the coacervate state, although α-helix was predominant after subsequent cross-linking of lysine side chains with genipin. Mutation of lysine residues to hydrophobic amino acids, tyrosine or alanine, leads to increased propensity for ß-structure and the formation of amyloid-like fibrils, characterized by thioflavin-T binding and transmission electron microscopy. These findings indicate that cross-linking domains are structurally labile during assembly, adapting to changes in their environment and aggregated state. Furthermore, the sequence of cross-linking domains has a dramatic effect on self-assembly properties of elastin-like polypeptides, and the presence of lysine residues in these domains may serve to prevent inappropriate ordered aggregation.

Elastin binding protein and FKBP65 modulate in vitro self-assembly of human tropoelastin.

Elastin is a protein that provides the unusual properties of extensibility and elastic recoil to tissues. Assembly of polymeric elastin into its final architecture in the extracellular matrix involves both self-aggregation properties of its monomeric precursor, tropoelastin, and interactions with several matrix-associated proteins that appear to act by modulating the intrinsic self-assembly of tropoelastin. Because of its highly nonpolar character and propensity to self-aggregate, it has been suggested that mechanisms limiting self-aggregation must also be present during the transit of tropoelastin through the cell prior to secretion. Both the elastin binding protein (EBP) and FKBP65 have been suggested to fulfill that role in the Golgi and endoplasmic reticulum compartments of the cell, respectively. However, details about the nature of the interactions between these proteins as well as about the mechanism by which they may act to limit self-aggregation are lacking. In this study, we demonstrate that both EBP and FKBP65 have strong binding affinities for tropoelastin, with the dissociation constant of EBP approximately 4-fold lower than that of FKBP65. Both proteins also modify the kinetics of self-assembly of tropoelastin in an in vitro system, consistent with a role in attenuating the premature intracellular self-aggregation of tropoelastin through a mechanism that limits the growth and maturation of aggregates. The ability of FKBP65 to modulate the self-assembly of tropoelastin is independent of its enzymatic activity to promote the cis-trans isomerization of proline residues in proteins.

Sequence and domain arrangements influence mechanical properties of elastin-like polymeric elastomers.

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self-assembly that aligns lysine residues for crosslinking. As a result, both the full-length monomer as well as elastin-like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full-length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self-assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full-length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties.

In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials.

BACKGROUND: Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown. METHODS: Small peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry. RESULTS: MS(2) data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations. CONCLUSIONS: The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials. GENERAL SIGNIFICANCE: The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin.

Molecular assembly and mechanical properties of the extracellular matrix: A fibrous protein perspective.

The extracellular matrix is an integral and dynamic component of all tissues. Macromolecular compositions and structural architectures of the matrix are tissue-specific and typically are strongly influenced by the magnitude and direction of biomechanical forces experienced as part of normal tissue function. Fibrous extracellular networks of collagen and elastin provide the dominant response to tissue mechanical forces. These matrix proteins enable tissues to withstand high tensile and repetitive stresses without plastic deformation or rupture. Here we provide an overview of the hierarchical molecular and supramolecular assembly of collagens and elastic fibers, and review their capacity for mechanical behavior in response to force. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

Polymorphisms in the human tropoelastin gene modify in vitro self-assembly and mechanical properties of elastin-like polypeptides.

Elastin is a major structural component of elastic fibres that provide properties of stretch and recoil to tissues such as arteries, lung and skin. Remarkably, after initial deposition of elastin there is normally no subsequent turnover of this protein over the course of a lifetime. Consequently, elastic fibres must be extremely durable, able to withstand, for example in the human thoracic aorta, billions of cycles of stretch and recoil without mechanical failure. Major defects in the elastin gene (ELN) are associated with a number of disorders including Supravalvular aortic stenosis (SVAS), Williams-Beuren syndrome (WBS) and autosomal dominant cutis laxa (ADCL). Given the low turnover of elastin and the requirement for the long term durability of elastic fibres, we examined the possibility for more subtle polymorphisms in the human elastin gene to impact the assembly and long-term durability of the elastic matrix. Surveys of genetic variation resources identified 118 mutations in human ELN, 17 being non-synonymous. Introduction of two of these variants, G422S and K463R, in elastin-like polypeptides as well as full-length tropoelastin, resulted in changes in both their assembly and mechanical properties. Most notably G422S, which occurs in up to 40% of European populations, was found to enhance some elastomeric properties. These studies reveal that even apparently minor polymorphisms in human ELN can impact the assembly and mechanical properties of the elastic matrix, effects that over the course of a lifetime could result in altered susceptibility to cardiovascular disease.

Coacervation of tropoelastin.

The coacervation of tropoelastin represents the first major stage of elastic fiber assembly. The process has been modeled in vitro by numerous studies, initially with mixtures of solubilized elastin, and subsequently with synthetic elastin peptides that represent hydrophobic repeat units, isolated hydrophobic domains, segments of alternating hydrophobic and cross-linking domains, or the full-length monomer. Tropoelastin coacervation in vitro is characterized by two stages: an initial phase separation, which involves a reversible inverse temperature transition of monomer to n-mer; and maturation, which is defined by the irreversible coalescence of coacervates into large species with fibrillar structures. Coacervation is an intrinsic ability of tropoelastin. It is primarily influenced by the number, sequence, and contextual arrangement of hydrophobic domains, although hydrophilic sequences can also affect the behavior of the hydrophobic domains and thus affect coacervation. External conditions including ionic strength, pH, and temperature also directly influence the propensity of tropoelastin to self-associate. Coacervation is an endothermic, entropically-driven process driven by the cooperative interactions of hydrophobic domains following destabilization of the clathrate-like water shielding these regions. The formation of such assemblies is believed to follow a helical nucleation model of polymerization. Coacervation is closely associated with conformational transitions of the monomer, such as increased ß-structures in hydrophobic domains and α-helices in cross-linking domains. Tropoelastin coacervation in vivo is thought to mainly involve the central hydrophobic domains. In addition, cell-surface glycosaminoglycans and microfibrillar proteins may regulate the process. Coacervation is essential for progression to downstream elastogenic stages, and impairment of the process can result in elastin haploinsufficiency disorders such as supravalvular aortic stenosis.