Probing fibronectin adsorption on chemically defined surfaces by means of single molecule force microscopy

Evangelos Liamas,Richard A Black,Paul A Mulheran,Ralph Wieneke,Zhenyu J Zhang,Owen R T Thomas,Robert Tampé

doi:10.1038/s41598-020-72617-z

Abstract

Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) and a quartz crystal microbalance (QCM) were respectively employed to probe interfacial characteristics of fibronectin fragment FNIII8–14 and full-length fibronectin (FN) on CH3–, OH–, COOH–, and NH2-terminated alkane-thiol self-assembled monolayers (SAMs). Force-distance curves acquired between hexahistidine-tagged FNIII8–14 immobilised on trisNTA-Ni2+ functionalized AFM cantilevers and the OH and COOH SAM surfaces were predominantly ‘loop-like’ (76% and 94% respectively), suggesting domain unfolding and preference for ‘end-on’ oriented binding, while those generated with NH2 and CH3 SAMs were largely ‘mixed type’ (81% and 86%, respectively) commensurate with unravelling and desorption, and ‘side-on’ binding. Time-dependent binding of FN to SAM-coated QCM crystals occurred in at least two phases: initial rapid coverage over the first 5 min; and variably diminishing adsorption thereafter (5–70 min). Loading profiles and the final hydrated surface concentrations reached (~ 950, ~ 1200, ~ 1400, ~ 1500 ng cm−2 for CH3, OH, COOH and NH2 SAMs) were consistent with: space-filling ‘side-on’ orientation and unfolding on CH3 SAM; greater numbers of FN molecules arranged ‘end-on’ on OH and especially COOH SAMs; and initial ‘side-on’ contact, followed by either (1) gradual tilting to a space-saving ‘end-on’ configuration, or (2) bi-/multi-layer adsorption on NH2 SAM.

Highlights

Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) and a quartz crystal microbalance (QCM) were respectively employed to probe interfacial characteristics of fibronectin fragment FNIII8–14 and full-length fibronectin (FN) on CH3, OH, COOH, and NH2terminated alkane-thiol self-assembled monolayers (SAMs)
To identify the contribution from each chemical species, force measurements were performed between the AFM tip and the SAMs surfaces at every step of the functionalization
Prior to examining FD curves of the chemically attached F NIII8–14 (‘gold-linker-FNIII8–14′) with the different SAM modified surfaces a hierarchical series of tests were conducted using bare (‘gold’), physisorbed FNIII8–14 (‘gold-FNIII8–14’), and trisNTA-EG3-C16-SH linker (‘gold-linker’) AFM cantilevers

Summary

Introduction

Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) and a quartz crystal microbalance (QCM) were respectively employed to probe interfacial characteristics of fibronectin fragment FNIII8–14 and full-length fibronectin (FN) on CH3–, OH–, COOH–, and NH2terminated alkane-thiol self-assembled monolayers (SAMs). SMFS has been applied in the study of FN’s mechanical stability and interactions with other molecules, notably integrins These investigations reveal FNIII modules containing the cell-binding site as weakest in the mechanical s ense[27,28,29,30] and that force-induced dissociation of the complex formed between α5β1 integrin and fibronectin fragment FNIII7–10 requires overcoming two activation barriers, indicating that integrin activation involves cooperative interaction of the F NIII10 RGD and FNIII9 PHSRN synergy sites[31]. At high FN challenge to the surface force-distance curves exhibited successive rupture events indicative of protein unfolding, while at low FN challenge FN partially denatured upon adsorption, generating force-distance curves lacking dominant pulling events

Methods

Results

Conclusion