Photosystem-II D1 protein mutants of Chlamydomonas reinhardtii in relation to metabolic rewiring and remodelling of H-bond network at Q(B) site

Photosystem II (PSII) reaction centre D1 protein of oxygenic phototrophs is pivotal for sustaining photosynthesis. Also, it is targeted by herbicides and herbicide-resistant weeds harbour single amino acid substitutions in D1. Conservation of D1 primary structure is seminal in the photosynthetic performance in many diverse species. In this study, we analysed built-in and environmentally-induced (high temperature and high photon fluency-HT/HL) phenotypes of two D1 mutants of Chlamydomonas reinhardtii with Ala250Arg (A250R) and Ser264Lys (S264K) substitutions. Both mutations differentially affected efficiency of electron transport and oxygen production. In addition, targeted metabolomics revealed that the mutants undergo specific differences in primary and secondary metabolism, namely, amino acids, organic acids, pigments, NAD, xanthophylls and carotenes. Levels of lutein, beta-carotene and zeaxanthin were in sync with their corresponding gene transcripts in response to HT/HL stress treatment in the parental (IL) and A250R strains. D1 structure analysis indicated that, among other effects, remodelling of H-bond network at the Q(B) site might underpin the observed phenotypes. Thus, the D1 protein, in addition to being pivotal for efficient photosynthesis, may have a moonlighting role in rewiring of specific metabolic pathways, possibly involving retrograde signalling

A whole cell optical bioassay for the detection of chemical warfare mustard agent simulants

Chemical warfare agents (CWAs) have become a crucial concern of the 20th century in the safety vision of the society and the environment, triggering a growing awareness for early alarm in case of terroristic attacks as well as preventing contamination. Herein, we present the development of a whole-cell optical bioassay for the detection of CWA simulants. In particular, the effects of the mustard agent simulants bis-2-chloroethyl amine and 2-chloroethyl ethyl sulphide on the unicellular green photosynthetic algae Chlamydomonas reinhardtii was studied, with the aim to unravel the response of the microorganism to the presence of these simulants and optimise the analytical conditions of the bioassay. Important variations in the growth, photosynthetic activity, and content of photosynthetic pigments were observed in the presence of the selected simulants. The algal response towards bis-2-chloroethyl amine and 2-chloroethyl ethyl sulphide in a concentration range between 0.2 and 2.5 mM was analysed, indicating a linear relationship in the measured dose-response curves and detection limits of 50 and 200 mu M, respectively. Interference studies demonstrated the suitability of the proposed optical bioassay to detect mustard agent simulants also in drinking water, a defenceless matrix in case of terroristic attack, where atrazine, copper, and arsenic could be present at safety limits. Very slight matrix effect was evidenced, with ratios of 1.04 and 0.86 for calibration curves of bis-2-chloroethyl amine and 2-chloroethyl ethyl sulfide in tap water samples with respect to curves of standard solutions. Recovery values of 104 +/- 15% and 97.5 +/- 6.5% for 1 mM and 2 mM of bis-2-chloroethyl amine and 93 +/- 16% and 105 +/- 5% for 1 mM and 2 mM of 2-chloroethyl ethyl sulfide were achieved. (c) 2017 Elsevier B.V. All rights reserved

Design and biophysical characterization of atrazine-sensing peptides mimicking the Chlamydomonas reinhardtii plastoquinone binding niche

The plastoquinone (Q(B)) binding niche of the Photosystem II (PSII) D1 protein is the subject of intense research due to its capability to bind also anthropogenic pollutants. In this work, the Chlamydomonas reinhardtii D1 primary structure was used as a template to computationally design novel peptides enabling the binding of the herbicide atrazine. Three biomimetic molecules, containing the Q(B)-binding site in a loop shaped by two alpha-helices, were reconstituted by automated protein synthesis, and their structural and functional features deeply analysed by biophysical techniques. Standing out among the others, the biomimetic mutant peptide, D1pepMut, showed high ability to mimic the D1 protein in binding both Q(B) and atrazine. Circular dichroism spectra suggested a typical properly-folded alpha-helical structure, while isothermal titration calorimetry (ITC) provided a complete thermodynamic characterization of the molecular interaction. Atrazine binds to the D1pepMut with a high affinity (K-d = 2.84 mu M), and a favourable enthalpic contribution (Delta H = -11.9 kcal mol(-1)) driving the interaction. Fluorescence spectroscopy assays, in parallel to ITC data, provided hyperbolic titration curves indicating the occurrence of a single atrazine binding site. The binding resulted in structural stabilisation of the D1pepMut molecule, as suggested by atrazine-induced cooperative profiles for the fold-unfold transition. The interaction dynamics and the structural stability of the peptides in response to the ligand were particularly considered as mandatory parameters for biosensor/biochip development. These studies paved the way to the set-up of an array of synthetic mutant peptides with a wide range of affinity towards different classes of target analytes, for the development of optical nanosensing platforms for herbicide detection