Biocatalytic activities of functionalized surfaces controlled by mechanical stimuli – BioStretch

Living systems are complex structures sensitive to stimuli coming from their microenvironment. Recently, many studies revealed the influence of mechanical stimuli on cell response. In particular, they focused on how cells induce cascades of signalisation and chemical responses through mechanosensing processes. These processes, called mechanotransduction, are based on conformational modifications of protein structures under external stretch. For example, in the case of the cell adhesion protein fibronectin, many "cryptic" binding sites buried within modules in the folded state have been identified. Under stretch, these adhesion sequences are unmasked and adhesion is thus promoted. Since the discovery of these biological processes, a new scientific domain emerged. It is focused on the study of modifications of affinity or reactivity of single molecules (proteins, enzymes) under mechanical stimuli. These results show that it should be possible to design new mechanical responsive surfaces based on these structural modifications of proteins under stretch. This area of study, in contrast to studies on single molecules, is still in its infancy. Some mechanical responsive materials have already been described. Yet, they focus only on colour changes with stretch. Moreover, these changes are rarely reversible. Our teams were the first to depict recently a surface that becomes enzymatically active under stretch and in a reversible way. This first example was based on films containing enzymes embedded inside polymers chains acting as a "barrier". Under stretch, this barrier becomes thinner and this allows an unmasking of the catalytic sites of the enzymes. However these complex architectures are hardly extendable to other systems.
The aim of the present project is to design new films and materials with an enzymatic activity that can be tuned by stretching them to variable degree. Stretching of the surfaces or the materials should induce a local stretching on the enzymes. We will thus develop a new class of materials mimicking, at a macroscopic level, the behaviour of cryptic proteins involved in mechanotransductive processes. To this aim, three systems will be studied :
1) silicone surfaces chemically modified and on which enzymes will be covalently grafted.
2) polyacrylamide gels containing covalently linked enzymes.
3) polyelectrolyte multilayer films partially cross-linked and deposited on silicone sheets. Enzymes will be covalently grafted to polyelectrolyte chains.

Two model enzymes will be used : alkaline phosphatase and beta-galactosidase. Fine detection of their activities through fluorescence emission is possible and these enzymes are commercially available at relatively low cost. They will be chemically modified by grafting spacer arms ending with acrylate or maleimide groups for the covalent linking with the surrounding structure (silicone, polyacrylamide gels or polyelectrolyte multilayers).
This new concept of material is based on the fact that stretching of the gels or silicone sheets should transmit the mechanical strain on enzymes and as a consequence should modify their structure and thus their enzymatic activity. Characterization of the stretching at a nanometric or molecular level also constitutes one of the challenges of this project. For this purpose, we will develop new molecules playing the role of mechano-molecular sensors. These molecules will be included in gels or graft on silicone sheets molecules containing two pyrene groups linked together by spacers. Under stretch, these two pyrenes should be splitted away and their fluorescence emission properties should be shifted.
Finally, the primary aims of this project is to lay the groundwork for a new class of materials that could be called "mechano-transductive materials".