Team:EPF-Lausanne/Project Abstract

What we want to do

Light-sensitive proteins can easily be found in nature, but they have not been studied a lot. In this project, our aim is to design and work with a fusion protein that would allow genetic regulation through light control. The primarily designed protein that we will start with is the LovTAP hybride protein. It has been designed by Strickland et al. and generously sent to us by Pr. Sosnick from the University of Chicago, department of Biochemistry and Molecular Biology.

The strategy that has been used to design this hybride protein was to fuse a light-sensitive domain (Lov in our case) with a regulatory domain (like the Trp operon). By joining these two domains so that they share a continuous helix, an allosteric switch could thus be created, the shared helix acting as a rigid lever arm. The idea is to allow transmission of the conformational change induced by light (on the light-sensitive domain) to the DNA-binding domain. This transmitted conformational change would then result in an increase or decrease of the regulatory domain's affinity for the DNA promoter site.

Because these helical contacts are among the domains, their disruption will cause a global shift in the conformation of the protein. Conversely a photoexcitation (which also changes the conformational ensemble of the protein) will shifts the relative affinity of the shared helix for each domains, thereby allowing a signal sensed by one domain to be allosterically propagated to the other domain.

The overal effect would thus be a genetic expression controlled by light! In other words, we are trying to built a light-controlled DNA-binding protein. There would be many applications to such a "switch" : it could kill bacteria at a certain point, stop their growth, or make them express specific proteins...

To improve the change induced by light (which is generally very unstable), we also plan a modeling part whose aim is to find which residue could be mutated in order to have a stable protein after the light induction. This would allow a better "ON/OFF" protein with less interferences.

Advantages

The advantages of such a system are :

• easy to use: we just need to apply light on the system
• precise in space: we can choose exactly the localization of the ray. This one can be as small as a laser beam (a fraction of mm), but can also cover a bigger surface, depending on the needs.
• precise in time: contrary to a ligand-based DNA-binding protein, we can clearly see the advantage of using this method. Indeed, as soon as the light is switched off, the shift will convert back, and the signal will be instantly stopped.
• reversible because it acts as a simple switch
• cheap: need to have a light beam only, there is no need to buy additionnal equipment to control the spread of the ligad for example.

Possible applications

Our system could be very useful for industry as for academic research, as a new tool for regulating the gene expression.
If we focus on the applications in industry:

• in bioreactors, used in biochemical engeneering. Currently, one main issue is that molecules added in bioreactors to perform a special reaction cannot be removed once in the medium, except for some long and very expensive procedures of filtration. A major advantage of our system is that it is easily reversible, just be switching on or off the light!

A big problem of bioreactor is to keep them sterile: it is not a problem as the light can be kept behind a sterile plastic wall, which only have to be transparent.