A common problem in medicine is the induction of “side effects”, a problem the public is all too familiar with in the form of the long lists narrated to you on television advertisements. Many of these problems arise from non-specific interactions of the medicine with other receptors unrelated to the problem a medicine attempts to solve. But if you could isolate your medicines, limiting them only to release in specific locations near the places you wanted to target, you could make medicines many times more efficient and less likely to have such effects. And where would you start if you wanted to isolate them? Well, first, you’d need tiny packages to hold them, and a way to open those packages on demand. This is the problem we set out to solve.
A common problem in medicine is the induction of “side effects”, a problem the public is all too familiar with in the form of the long lists narrated to you on television advertisements. Many of these problems arise from non-specific interactions of the medicine with other receptors unrelated to the problem a medicine attempts to solve. But if you could isolate your medicines, limiting them only to release in specific locations near the places you wanted to target, you could make medicines many times more efficient and less likely to have such effects. And where would you start if you wanted to isolate them? Well, first, you’d need tiny packages to hold them, and a way to open those packages on demand. This is the problem we set out to solve.
In nature, the catalytic enzymes responsible for ethanolamine metabolism are localized and attached to EutS inside the microcompartment through an inherent tagging mechanism utilizing EutC. EutC, one of the associated Eut proteins in vivo, is incorporated inside EutS microcompartments through a targeting sequence contained within the first 19 amino acids (EutC1-19) (3) . Therefore, we can fuse EutC1-19 to any protein for incorporation in EutS microcompartments. Last year, our team’s school proved this concept by fusing EutC1-19 to enhanced green fluorescent protein (EutC1-19-EGFP) and co-expressing EutC1-19-EGFP together with EutS resulting in the formation of green fluorescing microcompartments. In order to give us remote control over the assembly and disassembly of our compartments, our team utilized the photoconvertible compound azobenzene, incorporated into the structure of a phenylalanine for incorporation as a non-canonical amino acid (ncAA) called AzoPhe. Azobenzene has been shown to undergo a steric shift from cis to trans under the stimulation of light at the wavelength of ~360 nm (5). We reasoned that if AzoPhe was substituted at very specific residues in EutS, we could perhaps use this steric property to control formation and disassembly of compartments. We started similarly to how the CU team from last year had, using PyMOL and pyRosetta to pick sites within the EutS protein that might form the full superstructure under one conformation, but not the other. We used point mutations and a plasmid containing an engineered tRNA system to insert AzoPhe at the sites of our choosing.
We decided that last year’s system for testing our mutations required several refinements. First of all, Green Fluorescent Protein was not ideal for our system, as in order to visualize GFP we had to use a similar wavelength of light to that required to change AzoPhe’s conformation. Secondly, some fluorescent proteins would not make it inside the compartments, confounding our ability to say with certainty that they had successfully formed. We addressed both of these issues this year by moving to a new fluorescent protein called FusionRed. Adding both a EutC tag and a LVA protein degradation tag allows FusionRed to be isolated formed compartments and protected from protein degradation. Any FusionRed outside the compartment will be degraded and not contribute to background visualization. In addition to this, we utilized a protocol for isolating these compartments intact, as they eventually would need to be to deliver drugs and have plans for further improvements.
We decided that last year’s system for testing our mutations required several refinements. First of all, Green Fluorescent Protein was not ideal for our system, as in order to visualize GFP we had to use a similar wavelength of light to that required to change AzoPhe’s conformation. Secondly, some fluorescent proteins would not make it inside the compartments, confounding our ability to say with certainty that they had successfully formed. We addressed both of these issues this year by moving to a new fluorescent protein called FusionRed. Adding both a EutC tag and a LVA protein degradation tag allows FusionRed to be isolated formed compartments and protected from protein degradation. Any FusionRed outside the compartment will be degraded and not contribute to background visualization. In addition to this, we utilized a protocol for isolating these compartments intact, as they eventually would need to be to deliver drugs and have plans for further improvements.
