Project C2: Mechanically responsive sensors with cryptic sites

Principal Investigators: S. Schiller (Freiburg) / P. Schaaf (Strasbourg)
Collaborators: J. Rühe (Freiburg)
PhD Students: Chunyan Yao / Johan Longo

Current state of the research

Transformation of a mechanical stimulus, such as stretching, into a chemical or biological reaction is a process that is widely used by nature and is called transduction. It usually relies on conformational changes of proteins called cryptic site proteins. Fibronectin is one of the best known example of such proteins. These proteins possess active sites that are buried inside their core in the nonstretched state and become exhibited under stretching [1]. Mechano-transduction is an example of a wider and emerging field, namely that of chemical reactions governed by mechanical stress. This field has gained in interest over the last years, mainly through single molecule experiments [2]. On the other side, materials that respond chemically to a mechanical stress are still extremely rare. To our knowledge, only one macroscopic system, which becomes chemically active under stretching, has been reported so far, which leads to a product released into the contacting solution. It constitutes a first example of cryptic like material [3]. It is based on embedding enzymes in a polyelectrolyte multilayer capped by a barrier. Under stretching the barrier thins so that the enzymes are exposed to the solution and thus become active. While the systems describe the first cryptic-like surface, this concept can be hardly transferred to a large class of proteins, enzymes or smaller ligands.
 

Contributions of the participating groups

Three groups are involved in this project: The group of P. Schaaf (Strasbourg) has started to work on mechanically responsive films since three years. Recently, in collaboration with the group of J.C. Voegel (Strasbourg), they succeeded in creating the first cryptic like film. Moreover, with the group of V. Roucoules from Mulhouse they are working on the functionalization of silicone sheets through polymer plasma in order to create surfaces covered by brushes and responding to stretching. The group of S. Schiller is specialized in the synthesis of proteins modified specifically with synthetic amino-acids. These will be key components in our project. The group of J. Rühe is specialized in surface chemistry and modifications and in the synthesis of surfaces with polymer brushes. They have studied the interaction of surface-attached polymer brushes and networks with molecules of the environment and have shown that macromolecules in a contacting solution can penetrate the surface-attached layers only in an area at the interface where the segment density is low (‘penetration zone’).
 

Research project and collaborations

The goal of this project is to create surfaces and cryptic materials that under stretching exhibit sites allowing to interact specifically with receptors. We propose two novel ways to achieve this: (i) One way is to covalently couple cryptic site proteins or modules of cryptic site proteins onto a surface or in a hydrogel. Stretching the surface or the material should allow to exhibit the active sites rendering the surface or the material active.  (ii) The other way is to embed ligands into polymer brushes. Stretching should decrease the brush density and render the ligands accessible to their receptors (see figure).

The project is divided into two parts : (i) developing cryptic site surfaces and materials based on cryptic site proteins and (ii) developing mechanically responsive surfaces based on exhibition of ligands through brush surfaces. Part (i) of the project is based on a novel and powerful technique, namely the modification of proteins via the cotranslational incorporation of synthetic amino-acids that carry functional groups for covalent linkage to synthetic polyelectrolytes. As a model system, we will use the 1-2FN III domains that interact under stretching with the FN N-terminal 30 kDa fragment of the same protein. Since stretching is necessary in order to access the cryptic site, the protein must be covalently linked via synthetic amino acids to the stretchable substrate or the surrounding elastomer. We will replace one amino-acid on each fragment, 1FN III and 2FN III, by an amino acid that bears an alkyne group and a third amino-acid that bears a biotin moiety. The latter one is introduced in order to control the anchoring of the two reactive alkyne group containing fragments onto the surface (S. Schiller, Freiburg). The proteins will be anchored to silicone substrates modified by ethylene oxide moities which possess azide groups at one end (P. Schaaf, Strasbourg in collaboration with V. Roucoules, Mulhouse). The 1-2FN III domains will then be anchored on the surface through click chemistry. These proteins will also be inserted and covalently bound in poly(acrylamide) gels through a similar chemistry (P. Schaaf, Strasbourg in collaboration with P. Lavalle, Strasbourg). We will then investigate the interaction between these 1-2FN III domains and the FN 30 kDa fragment under stretching. This will be achieved by labeling fluorescently the FN 30 kDa fragment and by following the fluorescence on the stretched surface or material (P. Schaaf Strasbourg).

Part (ii) of the project will consist of creating surfaces covered by brushes or surface-attached polymer networks into which ligands, such as biotin or small proteins, are inserted. These ligands will be, at rest, be masked by the surface-attached polymers. The masking is an inherent property of the surface-attached polymer layer and macromolecules can only penetrate the outer perimenter of the surface-attached layer. Upon stretching of the substrate in xy-direction the penetration zone will increase significantly. To do this, methods have to be developed which allow to attach polymer networks or brushes to elastomeric substrates. The layers need to be characterized and the penetraction by contacting macromolecules will be studied (J. Rühe, Freiburg), the other based on plasma polymer modified silicone substrates (P. Schaaf, Strasbourg, in collaboration with V. Roucoules, Mulhouse). Under stretching, they should become unmasked due to a decrease of the brush density, allowing for the interaction with their receptors. Moreover, while returning to the unstretched state, the interactions should be broken so that one recovers the initial state of the surface.

This project is highly interdisciplinary and based on the complementary expertise of the involved groups, the Schiller group in the synthesis of the modified proteins, the Rühe group in the synthesis of brush surfaces, and the Schaaf group in mechano-responsive surfaces. Each of the three elements is essential for the successful completion of the project.


References
[1]V. Vogel, Annual Rev. Biophys. 35, 459 (2006).
[2]A. del Rio et al., Science 323, 638 (2009) .
[3]D. Mertz et al., Nature Mater. 8, 731 (2009).
[4]J. Wang et al., Angew. Chem. 46, 36, 6849 (2007).