Design of ionic tunable biosensors (CA-FP)

Natural Sciences
Leioa (Vizcaya)
1 Jan, 2021 to 30 Sep, 2021
1 Nov, 2020

General information

8 months
3-4 days per week

We sought to generate a genetically encoded calcium indicator (GECI) based on a motif of bacterial origin to overcome competition incidences induced by those using calmodulin. RTX (repeat in toxin) motif consists of several repeats of a GGxGxDxux (u: hydrophobic residue) motif. These repeats bind Calcium ions, forming a beta-roll motif, initially shown by the crystal structure of P. aeruginosa alkaline protease ((1): 1akl). The first six residues form a loop and the last three form a short beta-strand. RTX containing proteins are usually secreted to the external media where calcium concentration is high enough to promote folding. The motif apo-form is glycine-rich and highly charged and consequently disordered. Calcium binding is tightly linked to beta-roll folding and, differently to other calcium binding motives, due to the multiplicity of complementary sites, this occurs in a very narrow range of ligand concentration. The reference model in our study will consist of a single fusion protein in which two fragments of the FP would be located at the N- and C-terminus respectively separated by an optimized RTX motif. The high charge of the calcium-free linker should avoid spontaneous self-complementation of the two FP halves, thus minimizing background signals. Subtle increases of calcium concentration above the apparent dissociation constant (Kd) would switch fluorescence to its maximum. The number and sequence of the repeats will be altered to modulate affinity and cooperativity. Recent developments have significantly expanded the palette of self-associating spFPs and the combination of those with Ca2+-binding motives of different Kd (Ca2+) will provide an excellent arsenal for multi-color imaging. Moreover, the sorting of the biosensor to a given cellular type or compartment can be modulated by specifics tags

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Ca binding  fluorescent protein  motif design  
English: Independent User B2
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