Design of Perceval

Perceval, our sensor of the cellular ATP:ADP ratio, is a fusion protein that consists of a bacterial regulatory protein, GlnK1, and a modified green fluorescent protein (GFP). GlnK1 is a small trimeric protein with three nucleotide binding sites. These sites can bind either ATP or ADP - but only ATP produces a local conformational change that involves closure of the "T-loop" over the nucleotide binding site. To convert this conformational change to a fluorescent signal, we spliced a "circularly permuted" GFP variant (cpmVenus) into the T-loop.

A normal GFP has its N- and C-termini at the end of the β-barrel structure that encloses the fluorescent chromophore. Circularly-permuted GFP has the normal N- and C-termini linked together, and new N- and C-termini in the wall of the β-barrel, close to the chromophore. Conformational changes linked to these new termini can be coupled to fluorescent changes. This cartoon shows a single subunit of GlnK1 with Mg-ATP bound; the T-loop (dark blue) is linked to circularly permuted Venus (green).

We use directed evolution to tune the properties of a prototype sensor for use in living cells. Multiple rounds of semi-random mutagenesis - using either doped oligonucleotides or error-prone PCR - are combined with screening of the fluorescent response (in a programmable 96-well plate reader) to give the best response properties.

 

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