Oligomerisation and thermal stability of polyvalent integrin α5β1 ligands

Abstract

Please cite this article as: Michaela Kreiner, Olwyn Byron, Diana Domingues, Christopher F. van der Walle, Oligomerisation and thermal stability of polyvalent integrin α5β1 ligands, Biophysical Chemistry (2009Chemistry ( ), doi: 10.1016Chemistry ( /j.bpc.2009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 AbstractSynthetic oligomeric integrin α5β1 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, itis 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 µM, 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 α5β1 ligands to be more accurately interpreted, and has wider implications with respect to the utility of coiled coils as tools to facilitate protein oligomerisation.

Orientation and surface coverage of adsorbed fibronectin cell binding domains and bound integrin α5β1 receptors

“…Surfactant behaviour has been modelled on the basis of neutron reflectivity data, including the roughness parameter of the fitted interfaces. 41 Bead models of modular proteins of the known 3-D structure have been used to define their overall spatial arrangement 42 in the interpretation of analytical ultracentrifugation data 43 and neutron data. 44 We are currently acquiring data for FIII9 0 10 molecules specifically oriented at gold surfaces through a C-terminal thiol and intend to develop bead models for these simplified protein layers before moving to the FIII9-10/integrin layers described here.…”