Interdomain mobility and conformational stability of type III fibronectin domain pairs control surface adsorption, desorption and unfolding.
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 beta-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 alpha5beta1 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.
Pereira
P
P
Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, UK.
Kelly
S M
SM
Gellert
P R
PR
van der Walle
C F
CF
D000327:Adsorption; D019943:Amino Acid Substitution; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012822:Silicon Dioxide; D013499:Surface Properties
D005353:Fibronectins; D010446:Peptide Fragments; C501364:fibronectin type III like peptide, human; D012822:Silicon Dioxide