Thermodynamic and hydrodynamic properties of human tropoelastin. Analytical ultracentrifuge and pulsed field-gradient spin-echo NMR studies

• Published in: The Journal of biological chemistry 2001-07, Vol.276 (30), p.28042-28050
• Main Authors: Toonkool, P, Regan, D G, Kuchel, P W, Morris, M B, Weiss, A S
• Format: Article
• Language: English
• Subjects: Humans; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Proteins - chemistry; Proteins - metabolism; Software; Temperature; Thermodynamics; Tropoelastin - chemistry; Tropoelastin - metabolism; Water - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M103391200

Tropoelastin is the soluble precursor of elastin that bestows tissue elasticity in vertebrates. Tropoelastin is soluble at 20 degrees C in phosphate-buffered saline, pH 7.4, but at 37 degrees C equilibrium is established between soluble protein and insoluble coacervate. Sedimentation equilibrium studies performed before (20 degrees C) and after (37 degrees 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 approximately 2.2, suggesting a very highly expanded or asymmetric protein. Species 2 displayed a frictional ratio at 20 degrees C of 1.4 that increased to 1.7 at 37 degrees 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 spin-echo 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 degrees 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.