The 9th-10th type III fibronectin domain pair (9-10FNIII) has found widespread use as a biomimetic surface for cell adhesion. However, the effect of mutations to 9-10FNIII on its surface adsorption characteristics have not been investigated. Here we address this issue using total internal reflection fluorescence (TIRF) and circular dichroism spectroscopy, comparing two conformationally stable 9-10FNIII mutants against the wild type. Desorption of the 9-10FNIII mutants from the silica surface was minimal in comparison to desorption of 9-10FNIII. The extent and rate of protein desorption from silica was empirically matched by loss of secondary structure upon adsorption, with only the spectrum for 9-10FNIII showing extensive loss of the β-sandwich fold. For the proteins adsorbed to hydrophobic surfaces, only the CD spectra for the 9-10FNIII mutant constrained via an interdomain disulphide bridge showed similarity with the corresponding solution structure. Since the binding of 9-10FNIII to integrin α5β1 is highly dependent on the relative spatial arrangement of the two domains, we suggest that the observed differences in cell adhesion and spreading on wild type 9-10FNIII and mutants may in part be attributed to the extent of protein desorption and unfolding at the surface.
- type III fibronectin domains
- surface adsorption
- circular dichroism
- total internal reflection fluorescence spectroscopy
Pereira, P., Kelly, S. M., Gellert, P. R., & van der Walle, C. F. (2008). Interdomain mobility and conformational stability of type III fibronectin domain pairs control surface adsorption, desorption and unfolding. Colloids and Surfaces B: Biointerfaces, 64(1), 1-9. https://doi.org/10.1016/j.colsurfb.2007.12.015