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Structure-based design of novel Chlamydomonas reinhardtii D1-D2 photosynthetic proteins for herbicide monitoring.

ABSTRACT: The D1-D2 heterodimer in the reaction center core of phototrophs binds the redox plastoquinone cofactors, Q(A) and Q(B), the terminal acceptors of the photosynthetic electron transfer chain in the photosystem II (PSII). This complex is the target of the herbicide atrazine, an environmental pollutant competitive inhibitor of Q(B) binding, and consequently it represents an excellent biomediator to develop biosensors for pollutant monitoring in ecosystems. In this context, we have undertaken a study of the Chlamydomonas reinhardtii D1-D2 proteins aimed at designing site directed mutants with increased affinity for atrazine. The three-dimensional structure of the D1 and D2 proteins from C. reinhardtii has been homology modeled using the crystal structure of the highly homologous Thermosynechococcus elongatus proteins as templates. Mutants of D1 and D2 were then generated in silico and the atrazine binding affinity of the mutant proteins has been calculated to predict mutations able to increase PSII affinity for atrazine. The computational approach has been validated through comparison with available experimental data and production and characterization of one of the predicted mutants. The latter analyses indicated an increase of one order of magnitude of the mutant sensitivity and affinity for atrazine as compared to the control strain. Finally, D1-D2 heterodimer mutants were designed and selected which, according to our model, increase atrazine binding affinity by up to 20 kcal/mol, representing useful starting points for the development of high affinity biosensors for atrazine.

SUBMITTER: Rea G

PROVIDER: S-EPMC2786977 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Structure-based design of novel Chlamydomonas reinhardtii D1-D2 photosynthetic proteins for herbicide monitoring.

Rea Giuseppina G Polticelli Fabio F Antonacci Amina A Scognamiglio Viviana V Katiyar Prashant P Kulkarni Sudhir A SA Johanningmeier Udo U Giardi Maria Teresa MT

Protein science : a publication of the Protein Society 20091001 10

The D1-D2 heterodimer in the reaction center core of phototrophs binds the redox plastoquinone cofactors, Q(A) and Q(B), the terminal acceptors of the photosynthetic electron transfer chain in the photosystem II (PSII). This complex is the target of the herbicide atrazine, an environmental pollutant competitive inhibitor of Q(B) binding, and consequently it represents an excellent biomediator to develop biosensors for pollutant monitoring in ecosystems. In this context, we have undertaken a stud ...[more]

PMID: 19693932

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Project description:BackgroundThe cellular proteome and metabolome are underlying dynamic regulation allowing rapid adaptation to changes in the environment. System-wide analysis of these dynamics will provide novel insights into mechanisms of stress adaptation for higher photosynthetic organisms. We applied pulsed-SILAC labeling to a photosynthetic organism for the first time and we established a method to study proteome dynamics in the green alga Chlamydomonas reinhardtii, an emerging model system for plant biology. In addition, we combined the analysis of protein synthesis with metabolic profiling to study the dynamic changes of metabolism and proteome turnover under salt stress conditions.ResultsTo study de novo protein synthesis an arginine auxotroph Chlamydomonas strain was cultivated in presence of stable isotope-labeled arginine for 24 hours. From the time course experiment in 3 salt concentrations we could identify more than 2500 proteins and their H/L ratio in at least one experimental condition; for 998 protiens at least 3 ratio counts were detected in the 24 h time point (0 mM NaCl). After fractionation we could identify 3115 proteins and for 1765 of them we determined their de novo synthesis rate. Consistently with previous findings we showed that RuBisCO is among the most prominent proteins in the cell; and similar abundance and turnover for the small and large RuBisCO subunit could be calculated. The D1 protein was identified among proteins with a high synthesis rates. A global median half-life of 45 h was calculated for Chlamydomonas proteins under the chosen conditions.ConclusionTo investigate the temporal co-regulation of the proteome and metabolome, we applied salt stress to Chlamydomonas and studied the time dependent regulation of protein expression and changes in the metabolome. The main metabolic response to salt stress was observed within the amino acid metabolism. In particular, proline was up-regulated manifold and according to that an increased carbon flow within the proline biosynthetic pathway could be measured. In parallel the analysis of abundance and de novo synthesis of the corresponding enzymes revealed that metabolic rearrangements precede adjustments of protein abundance.

Dataset Information

Structure-based design of novel Chlamydomonas reinhardtii D1-D2 photosynthetic proteins for herbicide monitoring.

Publications

Structure-based design of novel Chlamydomonas reinhardtii D1-D2 photosynthetic proteins for herbicide monitoring.

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