Tropoelastin is the soluble precursor of elastin, the major component of the extracellular elastic fiber. Tropoelastin undergoes self-association via an inverse temperature transition termed coacervation, which is a crucial step in elastogenesis. Coacervation of tropoelastin takes place through multiple intermolecular interactions of its hydrophobic domains. Previous work has implicated those hydrophobic domains located near the center of the polypeptide as playing a dominant role in coacervation. Short constructs of domains 18, 20, 24, and a mutated form of domain 26 were largely disordered at 20°C but displayed increased order on heating that was consistent with the formation of -structures. However, their conformational transitions were not sensitive to physiological temperature in contrast to the observed behavior of the native domain 26. A polypeptide consisting of domains 17-27 of tropoelastin coacervated at temperatures above 60°C, whereas individually expressed hydrophobic regions were not capable of coacervation. We conclude that coacervation depends on the hydrophobicity of the molecule and, by inference, the number of hydrophobic domains. Tropoelastin mutants were constructed to contain a Pro 3 Ala mutation in domain 26, separate deletions of domains 18 and 26, and a displacement of domain 26. These constructs displayed unequal capacities for coacervation, even when they contained the same number of hydrophobic regions and comparable levels of secondary structure. Thus, the capability for coacervation is determined by contributions from individual hydrophobic domains for which function should be considered in the context of their positions in the intact tropoelastin molecule.
Tropoelastin is the soluble precursor of elastin that bestows tissue elasticity in vertebrates. Tropoelastin is soluble at 20°C in phosphate-buffered saline, pH 7.4, but at 37°C equilibrium is established between soluble protein and insoluble coacervate. Sedimentation equilibrium studies performed before (20°C) and after (37°C) coacervation showed that the soluble component was strictly monomeric. Sedimentation velocity experiments revealed that at both temperatures soluble tropoelastin exists as two independently sedimenting monomeric species present in approximately equal concentrations. Species 1 had a frictional ratio at both temperatures of ϳ2.2, suggesting a very highly expanded or asymmetric protein. Species 2 displayed a frictional ratio at 20°C of 1.4 that increased to 1.7 at 37°C, indicating a compact and symmetrical conformation that expanded or became asymmetric at the higher temperature. The slow interconversion of the two monomeric species contrasts with the rapid and reversible process of coacervation suggesting both efficiently incorporate into the coacervate. A hydrated protein of equivalent molecular weight modeled as a sphere and a flexible chain was predicted to have a frictional ratio of 1.2 and 1.6, respectively. Tropoelastin appeared as a single species when studied by pulsed field-gradient spinecho NMR, but computer modeling showed that the method was insensitive to the presence of two species of equal concentration having similar diffusion coefficients. Scintillation proximity assays using radiolabeled tropoelastin and sedimentation analysis showed that the coacervation at 37°C was a highly cooperative monomer-n-mer self-association. A critical concentration of 3.4 g/liter was obtained when the coacervate was modeled as a helical polymer formed from the monomers via oligomeric intermediates.Elastin forms a highly insoluble cross-linked extracellular matrix that is predominantly responsible for the elasticity of vertebrate tissue. The precursor of elastin, tropoelastin, is devoid of cross-links. Following secretion from the cell surface, tropoelastin undergoes coacervation, which is a process of selfassociation characterized by an inverse temperature transition (1). Tropoelastin is soluble in aqueous solution at room temperature in vitro, but upon raising the temperature to 37°C the solution becomes turbid as tropoelastin molecules associate to form large aggregates (2, 3). This process of coacervation results from multiple intermolecular interactions of the hydrophobic domains (3, 4). The tropoelastin coacervate is a thick viscoelastic phase that is not miscible with the overlying solution (5). On cooling to 20°C, the aggregates dissociate reversibly, and the solution turns clear. Alternating between hydrophobic domains in the protein are short sequences of amino acid residues that form the cross-linking domains (6). Coacervation may concentrate and align tropoelastin molecules prior to elastin formation via lysyl oxidase-mediated cross-linkage of the lysine residues that leads to a grow...
Tropoelastin is the soluble precursor of vertebrate elastin, but the gene is absent in lower eukaryotes, which are not organized into complex multicellular arrays. A synthetic gene encoding human tropoelastin with a high codon adaptation index was examined for expression in Saccharomyces cerevisiae. The expression of tropoelastin transcript increased ∼20-fold following induction. The native cytoplasm-expressed form of tropoelastin was not detected. In contrast, N-terminal fusion of the SUC2 invertase signal sequence of the yeast to tropoelastin led to the production of intracellular fulllength protein and enhanced its stability in vivo. There was no evidence of extracellular secretion of the protein. We conclude that eukaryotic cells are capable of expressing and retaining undegraded tropoelastin in the absence of a vertebrate-specific protein partner. Tropoelastin was directed to the yeast endoplasmic reticulum through the interim presence of the SUC2 signal peptide sequence.
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