Chromophore charge-state switching through copper-dependent homodimerisation of an engineered green fluorescent protein

Here, we have linked one of the most common protein-protein interaction events, homodimerisation, to an essential trace metal, copper, through engineering green fluorescent protein. Mutation of H148 to cysteine promotes the neutral phenolic chromophore in the monomer that excites predominantly at ∼400 nm. Homodimerisation <i>via</i> a copper-dependent disulphide bridge switches the chromophore to the charged phenolate that excites at ∼490 nm. The result is a ∼30 fold increase in the fluorescence emission ratio. Homodimerisation kinetics are further improved by optimising the sfGFP homodimer interface, generating the variant termed GFP-diS2. Structures of the monomeric and dimeric GFP-diS2 suggest that charge switching is through peptide bond flipping and the formation of a buried organised water network around the chromophore that spans the interface region. Fusion to a leucine zipper protein dimerisation element greatly increased the GFP-diS2 association rate making it a more effective copper sensor <i>in vitro</i> and <i>in vivo</i> with Cu(i) instigating the signal change quicker and at lower ion concentrations than Cu(ii). Thus, GFP-diS2 provides the framework for generating a sensitive genetically encoded copper sensor and will eventually be adapted to monitor one of the most important protein-protein interactions in biology, homo-oligomerisation.