Promoteur de la thèse: Elise Hennebert
Co-promoteur: Philippe Leclère
Résumé de la dissertation
Nature has always inspired humans to create new tools, materials and diverse systems. For 40 years, scientists have studied and mimicked nature to develop bio-inspired adhesive materials capable of bonding quickly and effectively underwater and in a saline environment. In this context, most studies have focused on the permanent adhesives of mussels, barnacles and tubeworms. Nevertheless, recently, increasing attention has been paid to temporary adhesion, which allows organisms such as echinoderms and flatworms to repetitively attach and detach from surfaces. In particular, the adhesive mechanism developed by the sea star Asterias rubens has been studied in depth. The adhesive secretion produced by this species is composed of 35 proteins, one of which, the Sea star footprint protein-1 (Sfp1), has been characterized. This large protein of 3853 amino acids is the second most abundant constituent of the secreted adhesive. It is secreted in the form of four subunits (Sfp1 Alpha, Beta, Delta and Gamma) displaying specific protein-, carbohydrate- and metal- binding domains. This molecular layout provides a relatively unexplored design paradigm for the development of novel bio-inspired adhesives. The aim of this thesis was therefore to produce and characterize multimodular recombinant adhesive proteins based on Sfp1.
The first part of this thesis focused on the production of recombinant proteins based on the sequence of Sfp1. The bacterium Escherichia coli was used to produce two fragments of Sfp1 comprising most of its functional domains: the C-terminal part of the Beta subunit (rSfp1 Beta C-term), comprising two discoidin domains and one calcium-binding EGF-like domain, and the complete Delta subunit (rSfp1 Delta), comprising one von Willebrand Factor type D (VWD) domain, one C8 domain, one trypsin inhibitor like cysteine rich (TIL) domain, and one galactose-binding lectin domain. Their analysis by native polyacrylamide gel electrophoresis and size exclusion chromatography showed that the proteins self-assemble and form oligomers and aggregates in the presence of NaCl. Moreover, they adsorb onto glass and polystyrene upon addition of Na+ and/or Ca2+ ions, forming homogeneous coatings or irregular meshworks, depending on the cation species and concentration. The coatings made of each of the two proteins had no cytotoxic effects on HeLa cells and even increased their proliferation.
In the second part, the nanomechanical properties of rSfp1 Beta C-term and rSfp1 Delta, and a mix of the two recombinant proteins, were characterized by Atomic Force Microscopy with an emphasis on functional characteristics such as adhesive properties, deformation and modulus of rigidity. The Peak Force Quantitative Nanomechanical Mapping (PF-QNM) mode was used to characterize the microstructure and nanomechanical properties of thin protein layers both in dry and wet (artificial seawater or Tris buffer supplemented with NaCl or CaCl2) conditions. In dry conditions, rSfp1 Beta C-term and the mix of rSfp1 Beta C-term and rSfp1 Delta coatings presented higher adhesion values than rSfp1 Delta alone and Bovine serum albumin, used as negative control. In artificial seawater, rSfp1 Beta C-term and rSfp1 Delta showed higher adhesion values than BSA and the mix of both proteins. Finally, viscoelastic properties (E’, E’’ and tan δ) of the protein layers were analysed at the nanoscale by a new technique named nano Dynamic Mechanical Analysis (nDMA). The results showed that all the coatings formed visco-elastic materials.
Finally, in the third part, the adsorption of rSfp1 Beta C-term and rSfp1 Delta was quantified and the parts of the proteins responsible for the adsorption ability were investigated. Based on the previous results, two conditions were selected to perform Surface Plasmon resonance experiments, in which the recombinant proteins form homogeneous coatings: 25 mM Tris buffer supplemented with 450 mM NaCl for rSfp1 Beta C-term, and 25 mM tris buffer supplemented with 150 mM Cacl2 for Sfp1 Delta. Five types of self-assembled monolayers (SAMs) were chemically tested: hydrophobic, hydrophilic, positive, negative and a protein-resistant poly(ethylene glycol) surface. rSfp1 Beta C-term exhibited significantly higher adsorption compared to fibrinogen (positive control) and BSA (negative control) on all SAM’s. rSfp1 Delta adsorbed significantly more on hydrophilic, negative and protein-resistant surfaces than both controls. Truncated recombinant rSfp1 Beta C-term proteins were produced in order to better characterize the adsorption behavior of this protein. The analysis of adsorption capacities on glass of these new recombinant proteins showed that two mechanisms are involved in rSfp1 Beta C-term adsorption: (1) one mediated by the EGF-like domain in presence of Ca2+, and (2) one mediated by the sequence of Sfp1 Beta with no homology with known functional domain in databases, in presence of Na+.
La défense publique de la thèse de Madame Mathilde Lefevre aura lieu le 20 avril 2021 à 16h par vidéo-conférence, le lien actif sera publié la veille sur ce site.