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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allostery ͉ DNA binding ͉ drug binding P rotein allostery, whereby binding of a ligand at one site in a protein alters the function of a distant site in the protein, is imperative for the regulation of most biological processes (1-3).Comparison of crystal structures of free and ligand-bound allosteric proteins has led to the commonly held view that allosteric proteins have the ability to assume two distinct conformations with different activities. Ligand binding is seen to act as the switch causing interconversion between these two conformational states. However, recent work indicates that ligand-induced changes in protein stability and dynamics that cannot be observed by classical structure determination may play a fundamental role in mediating allostery (1,4,5). To address the potential importance of ligand-induced protein stabilization in allosteric mechanisms, we have investigated the relationship of folding thermodynamics and allosteric mechanism in one of the best-characterized allosteric systems, the tetracycline (Tc) repressor (TetR) (6, 7).TetR is a homodimeric protein in which each monomer is comprised of an N-terminal DNA-binding domain and a Cterminal tetracycline-binding and dimerization (TBD) domain (Fig. 1). Interaction with the antibiotic, Tc, causes a large decrease in the DNA-binding affinity of TetR (8). Because the Tc-binding sites in TetR are Ͼ30 Å from the DNA-binding helices, an allosteric mechanism is responsible for this effect. The model to explain TetR allostery was constructed by comparing the X-ray crystal structures of TetR complexed with either Tc or its DNA operator. Tc binding was seen to induce a pendulum like movement of ␣-helix 4 that results in an increase of the distance between the DNA recognition helices of the two monomers (␣-helices 3 and 3Ј). This change in position of the recognition helices would prevent them from binding DNA because they could no longer fit into successive major grooves (9-13).Despite intensive study of this system, certain aspects of TetR function remain unexplained. If Tc binding were responsible for inducing a conformation of TetR that is incompatible with DNA binding, then the apo form of TetR would be expected to resemble its DNA-bound form. However, the distance between the DNA recognition helices in unliganded TetR is actually greater than that seen in the Tc-bound form of the protein, suggesting that the DNA-binding domains of apo-TetR must possess considerable flexibility to allow them to access the DNA-bound conformation (11). A variety of noninducible TetR mutants (ninTetR) are not subject to Tc-induced transcriptional derepression even though they still bind to Tc with WT affinity (14, 15). Another group of mutants, called reverse TetRs (revTetRs), bind DNA more tightly in the presence of Tc derivatives than in their absence (16)(17)(18)(19). A unified explanation for these phenotypes has been difficult to formulate because many of the amino acid substitutions causing them lie at positions that are not directly involved in drug binding o...

Background: Elastin is a polymeric protein providing extensibility and elastic recoil to tissues. Results: Cross-linking domain structure shifts from random coil to ␤-strand to ␣-helix during assembly of elastin matrix. Conclusion: Cross-linking domains have a previously unappreciated structural lability during assembly, which is highly susceptible to mutations of lysine residues. Significance: Identification of conformational transitions in cross-linking domains of elastin during self-assembly is essential for understanding the mechanisms of formation of the elastic matrix.

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 β-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 β-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.

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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.

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