Team:TU-Eindhoven/Description
Description
Phase separation as a toolbox
In eukaryotic cells, macromolecules and proteins are organized into membraneless organelles. These organelles are structures which are formed through phase separation, induced by multivalent protein-protein interactions (PPI). Due to the lack of a membrane surrounding these organelles, a rapid exchange of components with the cellular environment can take place. Furthermore, photobleaching experiments have shown recovery within seconds to minutes, indicating that the structure of these organelles is quite flexible.[1] [2]
Phase separation in nature
Examples of membraneless organelles are nucleolus, P bodies and stress granules. There is a wide range of the biological functions that membraneless organelles facilitate. The nucleolus is an example of a membraneless organelle and it produces ribosomes. P-bodies, also membraneless, are required for the translation and transport of mRNA (lack of P-bodies results in large amounts of untranslated mRNA). Next to the protein processing, protein storage and transport, the organelles are also important for signalling. An example is the activation of MAP kinase by the clustering of binding partners to a phosphorylated TCR.[3] The work of Banani et al.[4] has shown that multivalent scaffold molecules can assemble and phase separate to form membraneless organelles. These organelles are also able to recruit certain client molecules, by binding these client molecules to the free domains of the scaffold proteins. The recruitment of these client can be modified by adding stoichiometric excess of a scaffold protein or by varying the scaffold valency.
How will we use phase separation?
In our project, we will use the known interaction of proteins 14-3-3 and CT52 to form protein constructs which can phase separate by addition of a small molecule protein-protein interaction stabilizer, e.g. fusicoccin. This construct is expected to have a hydrogel-like structure. A difference in valency between the two constructs, 4:3 (CT52:14-3-3), should lead to free domains in the construct. At these free sites, client molecules can be recruited. This way, it will be possible to enhance the construct with bioactive functionalities. With the formation of this hydrogel-like structure, we hope to gain foundational insights, increase the (iGEM) toolbox, and help with proving the concept of making synthetic organelles. Furthermore, the gel can be useful in the lab by facilitating an enzyme-friendly/stimulating environment. The gel may also be used develop a sensor for (TEV) proteases.
