P F LoCascio et al.
University of Oxford
How do you choose chimeric pieces? Look for generalizable characteristics, e.g. mechanical transduction of a signal. They chose EnvZ-OmpR from E.coli. This is a generic system: phosphorylation triggered depending on the amount of osmotic pressure. They end up with a simple custom-designed biological circuit. Components are essentially isolated, so you can change one component at a time. They used the internal part with a different external component (the transmembrane response to osmotic pressure change). They used a conserved proline to change what was at the front of the construct.
Why a GPCR? They are a large class of pharmaceutical targets (>30%), and a working GPCR in a microbial system could be part of a molecular sensing tool (need to couple with WR signalling mechanism). Neurotensin is a GPCR from R.Norvegicus. When designing the hybrid, they need to couple the external signal with the internal transducer to activate the internal transcriptional response. HAMP domain needs a mechanical stimulus from linker peptid, to activate auto-phosphorylation. They know that the GPCR has some sort of twisting motion, but the 3d structure is not yet known. GPCRs have 7 transmembrane helices. There is an interesting "8th half-helix" sitting on the end of the 7th helix.
The sequencing was correct – all plasmids were verified correct. They plan to express plasmids into the reporter system, test ligand binding, and test dimerisation effect.
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