An in silico study of the Chlamydomonas reinhardtii D1-D2 proteins aimed at designing and producing site-directed mutants with increased affinity for biosensing applications

The D1-D2 heterodimer in the reaction centre core of phototrophs binds the redox plastoquinone cofactors, QA and QB, the terminal acceptors of the photosynthetic electron transfer chain in photosystem II. This complex is the target site of the commonly used herbicides such as triazines, uree, diazines and fenolic compounds, competitive inhibitors of QB binding site, and consequently it represents an excellent biomediator to develop biosensors for pollutant monitoring in ecosystems. In this context, we have undertaken an in silico study of the Chlamydomonas reinhardtii D1-D2 proteins aimed at designing mutants with increased affinity for atrazine as a model herbicide. A three-dimensional structure of the D1 and D2 proteins from Chlamydomonas reinhardtii has been modelled using the crystal structure of the highly homologous Thermosynechococcus elongatus proteins as template (Figure 1). A series of D1 and D2 mutants was then generated in silico and the atrazine binding affinity of the mutant proteins has been calculated. The D1-D2 heterodimer mutants were designed and selected which, according to our model, increase herbicide binding affinity by up to 20 kcal/mol, representing useful starting points for the development of high affinity biomediators. Then, three mutants were prepared by side-directed mutagenesis, characterized by fluorescence analysis and studied for the selective recognition ability by an equilibrium-adsorption method. The S268C and S264K biomediators showed outstanding hypersensitivity and resistance, respectively, to both triazine and urea classes of herbicides. When immobilised on a silicon septum, the biomediators were found to be stable for several months compared to the wild-type's half-life of 25 days at room temperature in the operational conditions. A portable optical biosensor for environmental monitoring was developed with limits of detection between 0.8 x 10-11 M and 3.0 x 10-9 M, depending on the target analyte. In addition, the biomediator reusability without obviously deterioration in performance was demonstrated in ten repeated cycles.