Team:Unimelb/Design

Design

Designing Bacterial Cell Surface Glutamate and GABA Sensors

Marvin et al. (2013; 2018) recently developed a series of fusion proteins that can be used to measure the concentration of glutamate at the surface of mammalian cells. These proteins (dubbed ‘iGluSnFr’s) consist of four functional parts: a signal sequence that directs the protein to the cell surface, an anchor that attaches the protein to the surface of the cell, a glutamate binding domain, and a green fluorescent protein (GFP) domain. GFPs emit green light at a specific wavelength and intensity, so by measuring the intensity of light at that wavelength researchers can determine the number of active GFPs in a sample. Most GFPs emit light continuously, but in its default state the GFP used by Marvin et al. is partially disassembled so that it cannot fluoresce. When the glutamate binding domain encounters glutamate, it will bind to it in a way that restructures the protein, pushing the disassembled part of the GFP back into place and causing it to begin fluorescing. So, the protein will only emit light while it is bound to glutamate. The greater the concentration of glutamate, the more time it will spend in the bound state. This means that measuring the intensity of light emitted by a cluster of iGluSnFrs allows researchers to accurately determine glutamate concentration.

Glut/GABA binding domain

Figure 1. Structure of iGluSnFr and iGABASnFr proteins. This figure was adapted from Marvin et al. (2019).

iGluSnFr has successfully been used to measure glutamate release by neurons. Marvin et al. (2019) have also developed a GABA detector using the same design but replacing the glutamate binding domain with a GABA binding domain. These proteins are ideal for providing the accurate measurement of glutamate and GABA that we need for our system, however the proteins designed by Marvin et al. are indented for expression in mammalian cells and will not function correctly in bacteria. The signal sequence and anchor that allow the sensor to be presented on the cell surface are both specific to mammalian cells. In order to use these sensors in our bacterial system we had to design a modified version of this protein containing parts that would present it on the surface of an E. coli cell.

To achieve this, we removed the eukaryotic localisation signal and transmembrane anchor domain and added an ice nucleation protein (INP) domain to the protein. INP are bacterial cell surface proteins. In their naturally occurring form, they consist of a transmembrane anchor and a repetitive section that facilitates the formation of ice crystals. However, this repetitive section can easily be replaced with other proteins that one wishes to express on the surface of a bacteria. The anchor of INPs is appealing because it acts as both a signal sequence to direct the nascent protein to cell surface, and as an anchor to retain the protein there. INPs are also well suited to our needs because, unlike other bacterial surface expression systems, INPs can successfully transport large proteins and to accommodate transport of proteins containing disulphide bonds, both categories that apply to our sensors.

Sequence schematic of iGluSnFr: Glut binding domain Sequence schematic of our modified iGluSnFr: Glut binding domain

As well as adding the INP anchor to or gene sequence, we included a flexible linker between the anchor and the beginning of the glutamate binding domain. We were concerned that if the binding domain were held too close to the surface of the cell it might not encounter glutamate frequently. The linker should provide enough flexibility for the glutamate binding pocket to face outward into the solution we are analysing.


Bibliography

  1. Marvin, J. S., Borghuis, B. G., Tian, L., Cichon, J., Harnett, M. T., Akerboom, J., ... & Orger, M. B. (2013). An optimized fluorescent probe for visualizing glutamate neurotransmission. Nature methods, 10(2), 162.

  2. Marvin, J. S., Scholl, B., Wilson, D. E., Podgorski, K., Kazemipour, A., Mueller, J. A., ... & Little, J. P. (2018). Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR. Nat. Methods, 15, 936-939.

  3. Marvin, J. S., Shimoda, Y., Magloire, V., Leite, M., Kawashima, T., Jensen, T. P., ... & Leidenheimer, N. J. (2019). A genetically encoded fluorescent sensor for in vivo imaging of GABA. Nature methods, 1.