Results of the VDE structural integrity during 10 ns molecular dynamics simulations.

<p>Representative results of the molecular dynamics simulations of VDE in water show the hydrogen bond, i.e. the distance between ND1-H121 and HH-Y214 (black trace) and the amount of water within the central hydrophobic barrel of VDE (grey trace, solvent contacts within 0.5 nm of Y214-HH). Opening of the barrel structure is required for dimerization and activation of VDE. In (A) protonation states of VDE were chosen as calculated using continuum electrostatics, B–F) the five target residues, i.e. with a pK_a within the activation range of VDE have been fixed in their protonated state, one at the time: D98 (B), D117 (C), H168 (D), D206 (E), D86 (F). G) same analysis was performed with all four candidate residues for pH dependent conformational change while in their protonated state.</p

Candidate residues responsible for pH dependent VDE conformational change.

<p>Superimposition of the VDE central domain structure (VDE_cd) at pH 7 and 5. The regions where structure organization is conserved in the two structures are shown in yellow while the ones showing differences are highlighted in red and blue for the inactive (VDE_pH7) and active (VDE_pH5) structure, respectively. The second monomer of the active dimeric structure is shown in grey to visualize the dimerization interface. Major rearrangements upon activation involve loops L1, L3, L5, L7. Putative residues involved in the pH dependent conformational change (D86, D98, D117, D206, H168) are shown as green sticks. A) Top view of the monomers. B) same as A after x axis rotation of 90°.</p

Electrostatic interaction node maps and position of H121-Y214 hydrogen bond.

<p>Electrostatic interaction node maps generated from the results of the multi conformer continuum electrostatic calculations for A) the VDE_pH7 and B) the VDE_pH5. Acids and bases are shown as red and blue, respectively. The width of the edge is scaled according to the strength of the interaction. Green and red edges indicate stabilizing or de-stabilizing interactions, respectively. Electrostatic interaction node maps of the VDE_pH5 show residues of chain B opaque. A contact map of the two subunits is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035669#pone.0035669.s002" target="_blank">Figure S2</a>. Interaction are only shown if the absolute electrostatic interaction is greater than 0.4 kcal mol⁻¹. The graph densities are 0.059 and 0.029, for VDE_pH7 (A) and VDE_pH5 (B) node maps, respectively. Interaction cutoff is 0.2 kcal mol⁻¹. Major interaction hubs, representing <i>k-cores</i> of 4 or 7 and 6 in the VDE_pH7 (A) or VDE_pH5 (B) form, respectively, are highlighted with yellow circles.</p

Position of H121-Y214 hydrogen bond in VDE_pH7 structure.

<p>A) Top view of the inactive monomer, shown as grey cartoons. H121 and Y214 are shown as red sticks. B) Same as C after x axis rotation of 90° rotation. A part of the β-barrel is not shown for the sake of clear visualization of the H121–Y214 hydrogen bond.</p

Enzyme activity of VDE variants compared to WT.

<p>The enzyme activity of all generated mutants is reported, as determined with a spectroscopic method exploiting the different absorption of violaxanthin and zeaxanthin at 502 nm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035669#pone.0035669-Yamamoto1" target="_blank">[24]</a>. Activity is expressed as % of WT control sample, together with SD (n = 5). The protein amount employed for enzymatic essays was verified to be equivalent for WT and mutants by Western blotting using specific antibodies.</p

Evaluation of lipid productivity.

<p>Dependence of lipid production on illumination intensity. Productivity values with constant light (black squares) compared with the ones with light flashes of 350 and 1200 µE m⁻² s⁻¹ at various frequencies.</p

Algal growth kinetics under pulsed light.

<p>A–B) <i>Nannochloropsis</i> growth curves in pulsed light of differing intensity and frequency, 1200 µE m⁻² s⁻¹ (10, 5, 1 Hz, respectively in red, blue and green, A) and 350 µE m⁻² s⁻¹ (10 and 30 Hz in red and blue, B). Kinetics with 120 µE m⁻² s⁻¹ continuous light reported for comparison (black). C) Growth rate (columns) and cellular concentration after 8 days of growth (red squares) extrapolated from curves in A–B. Values with 120 and 1000 µE m⁻² s⁻¹ constant illumination from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038975#pone-0038975-g001" target="_blank">Figure 1</a> reported for comparison. D) Cell concentration in C reported normalized to integrated light intensity. 1200-10, 5, 1 Hz and 350-10, 30 Hz reported in dark blue, pink, light blue, red and green respectively.</p

<i>Nannochloropsis salina</i> growth under different light intensities in a flat-bed photobioreactor.

<p>A) Growth kinetics of algae exposed to differing light intensities from 5 to 1000 µE m⁻² s⁻¹. Data with 5, 50, 120, 150, 250, 350 and 1000 µE m⁻² s⁻¹ shown in light blue, black, red, green, dark blue, pink and yellow, respectively. B) Growth parameters determined from curves in A, specific growth rate (black squares) and cellular concentration after 8 days of growth (red circles). C) Cellular concentrations reported in B normalized to light intensity: this may be used as approximate estimate of biomass production, as no significant deviation of cell size or DW/cell ratio was observed.</p

Acclimation response in cells grown in continous vs. pulsed light.

<p>Cells grown under differing light intensities, either continuous or pulsed, compared with their Chl content per cell (black) and Chl/Car ratio (red), parameters indicating activation of acclimation response to pulsed light conditions.</p

Dependence of photosynthetic efficiency (Fv/Fm) on illumination conditions.

<p>Fv/Fm values at end of exponential phase, compared between cells grown under continuous illumination of differing intensity (black squares) and pulsed light of differing intensity and frequency, 1200 µE m⁻² s⁻¹ (10, 5, 1 Hz, red, green and blue circles), 350 µE m⁻² s⁻¹ (10 and 30 Hz, pink and light blue diamonds). Cells at 5 µE m⁻² s⁻¹ were too dilute to provide reliable results.</p