Presentation_1_3D Structures of Plant Phytochrome A as Pr and Pfr From Solid-State NMR: Implications for Molecular Function.PDF

<p>We present structural information for oat phyA3 in the far-red-light-absorbing (Pfr) signaling state, to our knowledge the first three-dimensional (3D) information for a plant phytochrome as Pfr. Solid-state magic-angle spinning (MAS) NMR was used to detect interatomic contacts in the complete photosensory module [residues 1–595, including the NTE (N-terminal extension), PAS (Per/Arnt/Sim), GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) and PHY (phytochrome-specific) domains but with the C-terminal PAS repeat and transmitter-like module deleted] auto-assembled in vitro with ¹³C- and ¹⁵N-labeled phycocyanobilin (PCB) chromophore. Thereafter, quantum mechanics/molecular mechanics (QM/MM) enabled us to refine 3D structural models constrained by the NMR data. We provide definitive atomic assignments for all carbon and nitrogen atoms of the chromophore, showing the Pfr chromophore geometry to be periplanar ZZEssa with the D-ring in a β-facial disposition incompatible with many earlier notions regarding photoconversion yet supporting circular dichroism (CD) data. The Y268 side chain is shifted radically relative to published Pfr crystal structures in order to accommodate the β-facial ring D. Our findings support a photoconversion sequence beginning with Pr photoactivation via an anticlockwise D-ring Za→Ea photoflip followed by significant shifts at the coupling of ring A to the protein, a B-ring propionate partner swap from R317 to R287, changes in the C-ring propionate hydrogen-bonding network, breakage of the D272–R552 salt bridge accompanied by sheet-to-helix refolding of the tongue region stabilized by Y326–D272–S554 hydrogen bonding, and binding of the NTE to the hydrophobic side of ring A. We discuss phyA photoconversion, including the possible roles of mesoscopic phase transitions and protonation dynamics in the chromophore pocket. We also discuss possible associations between structural changes and translocation and signaling processes within the cell.</p

Phylogenetic tree inferred using the Maximum likelihood method of 21 amino acid sequences of <i>Exiguobacterium</i> spp. closely related with E17R.

<p>The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.</p

Multiple protein alignment of PR from <i>Exiguobacterium</i> sp. S17 (E17R), green-light absorbing proteorhodopsin from <i>Exiguobacterium sibiricum</i> (ESR) and blue-light absorbing proteorhodopsin from the uncultured gamma-proteobacterium “Hot 75m4” (BPR).

<p>Residues shared between all the protein variants are marked with asterisks. Single amino acid residue at position 106 (BPR numbering) that functions as a spectral tuning switch and accounts for most of the spectral difference between the two pigment families is highlighted in light grey. Primary proton acceptor and donor are highlighted in dark grey (D86) and with a frame (K97), respectively. The Schiff base (K232 for ESR, K226 for E17R) is indicated by a diamond. Residues differing between both green-PRs are indicated with arrows. The seven transmembrane α-helices are indicated with blue bubbles.</p

Transient absorbance changes of E17R.

<p>A. The kinetics of the absorbance changes are shown for selected wavelengths (399 nm, 517 nm and 598 nm). B. The decay-associated spectra are depicted as obtained from the global fit. The corresponding time constants are given in the figure.</p

Electrometric record of E17R (in 20 mM Hepes, pH 7.4) with the BLM system.

<p>A. Transient currents and B. after the addition of the protonophore FCCP. Black bars indicate illumination with a 75 W XBO long-pass filtered at >495 nm. The grey bar shows the additional excitation of the M-state (>380 nm).</p

Absorption spectrum of E17R in 20 mM Hepes, 100 mM NaCl, 0.03% DDM.

<p>The inset shows the difference spectrum after bleaching with hydroxylamine. The extinction coefficient was calculated using the absorbance of the oxime product ΔA(oxime) in comparison to the bleached rhodopsin absorbance ΔA(Rh) as reference.</p

FPALM images of <i>E. coli</i> cells expressing wt YtvA.

<p>FPALM images of <i>E. coli</i> cells expressing wt YtvA soaked in isotonic buffer (A) and under de-hydrated conditions (B). Intracellular distribution seems similar for the system in both hydration states, the protein remains widespread over the cell body with some major aggregates at the cell wall. Experimental conditions for the de-hydrated state: Intensity: 0.2 W/cm² at 405 nm and 0.05 kW/cm² at 488 nm; for the hydrated state: Intensity: 1–2 W/cm² at 405 nm and 0.2 kW/cm² at 488 nm; frame rate: 30 Hz and total number of collected frames: 20000. Scale bar: 1 µm.</p

Dark recovery kinetics of <i>E. coli</i> colonies expressing selected YtvA mutants.

<p><b>A.</b> Dark relaxation of YtvAL to YtvAD after 5 minutes illumination with LED465 of <i>E. coli</i> colonies expressing the R63K mutant of YtvA. <b>B.</b> Dark relaxation of YtvAL to YtvAD after 5 minutes illumination with LED465 of <i>E. coli</i> colonies expressing Q123N YtvA. Green curves were measured for colonies smeared on a glass coverslip, red curves were obtained for colonies soaked in an isotonic buffer. Black curves are the best fits to stretched exponential decays. Parameters are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107489#pone-0107489-t001" target="_blank">Table 1</a>.</p

Dark recovery kinetics of dried YtvA.

<p><b>A.</b> Absorption spectrum for YtvA molecules air-dried on a quartz plate before (black) and after (red) photoconversion with LED465. The green curve reports the absorption spectrum at t = 24660 s at 25°C. After 24 hours the YtvAD spectrum is fully recovered (not shown). <b>B.</b> Dark recovery kinetics of YtvAL to YtvAD followed through the absorbance at 475 nm. The red solid line is the best fit with a double stretched exponential relaxation, with τ = 600±400 s and β = 0.6±0.2 (16%) and τ = (3.7±0.3)×10⁴s and β = 1.0±0.2 (84%).</p

Dark recovery kinetics of YtvAL to YtvAD.

<p><b>A.</b> Dark recovery of YtvAL to YtvAD in <i>E. coli</i> colonies expressing YtvA deposited on a glass coverslip, after 5 minutes illumination with LED465, monitored through fluorescence emission of YtvAD. The blue bars indicate illumination periods with LED465, black bars indicate that colonies were kept in the dark. T = 25°C. <b>B.</b> Dark relaxation of YtvAL to YtvAD, as monitored by the recovery of fluorescence emission after switching off LED465. Blue, buffered YtvA solution; red, <i>E. coli</i> colonies expressing YtvA soaked in 10 mM phosphate buffer solution, containing 0.9% NaCl (W/V), pH = 7.4; green, <i>E. coli</i> colonies expressing YtvA smeared on a glass coverslip. T = 25°C. Black solid lines correspond to fits with a stretched exponential relaxation (<i>E. coli</i> colonies expressing YtvA smeared on a glass coverslip) or with an exponential decay (YtvA solution and bacteria soaked in a buffer). <b>C.</b> Dark relaxation of YtvAL to YtvAD after 5 minutes illumination with LED465 of <i>B. subtilis</i> colonies expressing YtvA. Green curves were measured for colonies smeared on a glass plate, red curves were obtained for colonies soaked in an isotonic buffer. Black curves are the best fits with an exponential decay (colonies soaked in a buffer) or a stretched exponential decay (colonies smeared on a glass plate). Fitting parameters are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107489#pone-0107489-t001" target="_blank">Table 1</a>.</p