title: Oligomerisation and thermal stability of polyvalent integrin alpha5beta1 ligands.
abstract: Synthetic oligomeric integrin alpha5beta1 ligands, specifically immobilised to surfaces, facilitate increased fibroblast cell spreading compared with that associated with the monomer. These ligands consist of a N-terminal fibronectin domain pair, a spacer and a di-, tri- or tetrameric coiled coil. However, it is not yet clear what effect fusion of the fibronectin domains has on the predicted oligomerisation of the coiled coils. Using analytical ultracentrifugation we show that the predicted tetrameric and trimeric coiled coils facilitate a corresponding ligand oligomerisation with half-dissociation at 0.7 and 0.2 microM, respectively. In contrast, the predicted dimeric coiled coil formed both dimers and trimers. Under non-reducing conditions, the unique C-terminal thiol-facilitated inter-oligomer dimerisation of the trimeric species, generating hexameric ligands. Disulphide bonding also increased helical stability during thermal unfolding. The work allows the cellular response to these clustered integrin alpha5beta1 ligands to be more accurately interpreted, and has wider implications with respect to the utility of coiled coils as tools to facilitate protein oligomerisation.
authors.affiliation: Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, UK.
authors.forename: Michaela
authors.initials: M
authors.lastname: Kreiner
authors.forename: Olwyn
authors.initials: O
authors.lastname: Byron
authors.forename: Diana
authors.initials: D
authors.lastname: Domingues
authors.forename: Christopher F
authors.initials: CF
authors.lastname: van der Walle
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003198:Computer Simulation; D019281:Dimerization; D005353:Fibronectins / Q000737:chemistry; D039081:Integrin alpha5beta1 / Q000737:chemistry*; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D013696:Temperature; D014461:Ultracentrifugation
chemical_list: D005353:Fibronectins; D039081:Integrin alpha5beta1; D008024:Ligands