Design of stimuli-responsive OmpF-conjugates as biovalves for nano-reactors

Without compartmentalisation, there would be no life, as cells require spatial organization to perform all metabolic processes to sustain it. Compartmentalisation, or physical separation of biological reactions requires defined reaction spaces and active control over all molecules that enters or leaves them. Nature utilises for this purpose cell membranes and a plethora of different membrane proteins, which act as tunnels allowing and controlling molecular flow across the membrane. In a similar manner mimicking nature, by molecular self-assembly, allows us to produce artificial cells as simplified models for better understanding specific parts of cells. Our knowledge allows us today to go a step further, as we can use natural or artificial enzymes and trans-membrane channel proteins to create artificial organelles (nanoreactors) capable of supporting selective reactions and replacement the deficient structures or organelles. That is why the nanoreactors are gaining more and more interest for specific applications in the field of nano-medicine, analytics and advanced functional materials.
To achieve the desired compartmentalization, it was needed to advance from the artificial assemblies, with passive membrane transport, based on permanently open pores. First attempts in this direction are represented by the design of single stimuli triggered transport via structures capable of opening upon acidification or reduction. The second step will be to have reversible triggered transport structures, like cells have. To have reliable artificial replacements of the dysfunctional cell structures is needed.
This thesis, is achieving this necessary step by the development of a biovalve which can open and close on demand in a similar manner that the trans-membrane proteins work. This is a significant advance in the design of new cell-like polymeric compartmentalised structures with sustainable specific function. This new system can be switched on and off by demand, theoretically endlessly, opening new ways in the development of artificial structures to replace the non-functional ones in living cells.
This biovalve was obtained by modifying trans-membrane proteins (Omp F) capable of passive transport of a range of molecules, with selected pH sensitive peptides capable of opening and closing its pore. This new biovalve functionality was tested by reconstituting it in an artificial membrane of a nanoreactor that delimits an inner empty space in which selected enzymes were entrapped. The specific substrate for the entrapped enzyme was added on top of the assembled nanoreactors mixture, outside of the closed polymeric compartments. At physiological pH the biovalve opens, and the substrate molecules diffuse passively in the inner space of the nanoreactor entrapping the enzyme molecules. The enzymatic reaction takes place and the (fluorescent) product formation is monitored. At low pH the biovalve closed blocking the access of the substrate molecules and no fluorescent product can be detected.