Molecular Orientation of Tropoelastin is Determined by Surface Hydrophobicity Anton P. Le Brun, † John Chow, ‡ Daniel V. Bax, ‡ Andrew Nelson, † Anthony S. Weiss, ‡ and Michael James* ,†,§ † Bragg Institute, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, NSW 2232, Australia ‡ School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia § School of Chemistry, University of New South Wales, Kensington, NSW 2052, Australia * S Supporting Information ABSTRACT: Tropoelastin is the precursor of the extracellular protein elastin and is utilized in tissue engineering and implant technology by adapting the interface presented by surface-bound tropoelastin. The preferred orientation of the surface bound protein is relevant to biointerface interactions, as the C-terminus of tropoelastin is known to be a binding target for cells. Using recombinant human tropoelastin we monitored the binding of tropoelastin on hydrophilic silica and on silica made hydrophobic by depositing a self-assembled monolayer of octadecyl trichlorosilane. The layered organization of deposited tropoelastin was probed using neutron and X-ray reflectometry under aqueous and dried conditions. In a wet environment, tropoelastin retained a solution-like structure when adsorbed on silica but adopted a brush-like structure when on hydrophobized silica. The orientation of the surface-bound tropoelastin was investigated using cell binding assays and it was found that the C-terminus of tropoelastin faced the bulk solvent when bound to the hydrophobic surface, but a mixture of orientations was adopted when tropoelastin was bound to the hydrophilic surface. Drying the tropoelastin-coated surfaces irreversibly altered these protein structures for both hydrophilic and hydrophobic surfaces. ■ INTRODUCTION Elastin is the extracellular matrix protein primarily responsible for imparting elasticity to tissues that experience extension and contraction. In humans, elastin forms over 90% of the elastic fibers found in tissues such as arteries, lung and the skin dermis. 1 Elastin is stable in adult humans with a very low turn- over rate, 2,3 and while its regeneration can be stimulated in adult organisms, they are generally ill-equipped to do so. 4,5 Synthetic elastin is therefore an important biomaterial for tissue engineering and repair. 6 Elastin is assembled from its soluble precursor, tropoelastin, a 60 kDa monomer protein that assembles into elastin fibers by a reversible self-aggregation process called coacervation. 6 Tropoelastin is composed of alternating hydrophilic and hydro- phobic domains. Hydrophilic domains are characterized by their high lysine and alanine contents and play roles in cross- linking processes. 7 The hydrophobic domains are enriched by nonpolar residues valine, glycine, and proline that typically occur in repeating motifs. While the tertiary structure of tropoelastin has not yet been definitively determined, analysis of individual domains indicates secondary structures such as poly- proline II (PPII) and disordered structure. 8,9 The C-terminus region of tropoelastin has been shown to play a key role in the assembly into elastin fibers. 10 Characteristic features of this region include the only two cysteine residues in the protein, which forms a disulfide bond and its termination in a positively charged RKRK sequence. 7 Due to the inherent insolubility and flexibility of elastin, 11-13 structural analysis has focused on studies of tropoelastin in solution; 13,14 while electron microscopy of deposited tropo- elastin has given only limited information on the shape of the protein. 15 Small-angle X-ray scattering (SAXS) corroborated with small angle neutron scattering (SANS) of tropoelastin and its subfragments revealed that tropoelastin adopts a defined nanostructure that consists of specific modules that specialize in elasticity and dermal fibroblast interactions. 16 Atomic force microscopy and photoelectron spectroscopy studies of elastin- like peptides immobilized to silica showed that the structure of adsorbed chains differs from that in solution, but as they correspond to small parts of the protein, their relevance to tropoelastin is doubtful. 17,18 Surface-bound tropoelastin is being tested in tissue engineer- ing and implant technology 19 bound to hydrophobic surfaces such as polyethylene glycol terephthalate (PTFE), 20 hydro- philic surfaces such as oxidized polystyrene 21 and graded metal surfaces. 22,23 Three-dimensional scaffolds made by electro- spinning and in the form of hydrogels 24-26 support cell growth, while tropoelastin has been covalently bonded to plasma polymer coated stainless steel surfaces for use in coronary stents. 27,28 Elastin-coated surfaces have been shown to affect Received: October 9, 2011 Revised: December 5, 2011 Published: December 17, 2011 Article pubs.acs.org/Biomac © 2011 American Chemical Society 379 dx.doi.org/10.1021/bm201404x | Biomacromolecules 2012, 13, 379-386