Thermodynamic and Hydrodynamic Properties of Human Tropoelastin

Abstract

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...

Charge‐Tunable Autoclaved Silk‐Tropoelastin Protein Alloys That Control Neuron Cell Responses

“…Supplemental Figure S2 shows the hydrodynamic radius (Rh) vs. intensity distribution of all silk-tropoelastin samples in 0.1-wt% solution (SE0, SE10, SE25, SE50, SE75, SE90, and SE100). Notably, scattered light at a hydrodynamic radius (Rh) of around 6 nm with a predicted molecular weight of 59±12 kDa for sample SE0 is due to pure tropoelastin monomers in solution, as previously reported 35, 36 . In contrast, pure silk (SE100) showed strong intensity of scattered light at Rh of 57.1±9.1 nm and a predicted molecular weight of 427±84 kDa, indicating that silk monomers and some small silk aggregates were formed in solution 12, 22 .…”

Hydrophobic Domains of Human Tropoelastin Interact in a Context-dependent Manner

“…Results from previous experiments proposed a nucleationcondensation model for coacervation of full-length tropoelastin SHEL⌬26A at 37°C (21). This model, together with the putative dominant role for domains 18, 20, 24, and 26 during coacervation, suggested that even though SHEL17-27 did not coacervate at 37°C, it may have been able to self-polymerize as part of the nucleation phase prior to coacervation.…”

“…These results indicated a greater capacity for intermolecular interactions by SHEL17-27 compared with individual hydrophobic domains. It is possible that at a sufficiently high concentration, SHEL17-27 could form a high molecular weight oligomer that may be representative of the nucleating species for the nucleation-condensation model described for tropoelastin (21). These oligomers, however, may not necessarily be dimers and do not account for the coacervation of full-length tropoelastin under physiologically relevant conditions at 37°C.…”

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

“…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)(4)(5) and critically, retains substantial backbone hydration and flexibility even when assembled (6) and cross-linked into mature, polymeric elastic arrays (7)(8)(9)(10)(11)(12)(13).…”