Green Proteorhodopsin Reconstituted into Nanoscale Phospholipid Bilayers (Nanodiscs) as Photoactive Monomers

Over 4000 putative proteorhodopsins (PRs) have been identified throughout the oceans and seas of the Earth. The first of these eubacterial rhodopsins was discovered in 2000 and has expanded the family of microbial proton pumps to all three domains of life. With photophysical properties similar to those of bacteriorhodopsin, an archaeal proton pump, PRs are also generating interest for their potential use in various photonic applications. We perform here the first reconstitution of the minimal photoactive PR structure into nanoscale phospholipid bilayers (nanodiscs) to better understand how protein–protein and protein–lipid interactions influence the photophysical properties of PR. Spectral (steady-state and time-resolved UV–visible spectroscopy) and physical (size-exclusion chromatography and electron microscopy) characterization of these complexes confirms the preparation of a photoactive PR monomer within nanodiscs. Specifically, when embedded within a nanodisc, monomeric PR exhibits a titratable p<i>K</i>_a (6.5–7.1) and photocycle lifetime (∼100–200 ms) that are comparable to the detergent-solubilized protein. These ndPRs also produce a photoactive blue-shifted absorbance, centered at 377 or 416 nm, that indicates that protein–protein interactions from a PR oligomer are required for a fast photocycle. Moreover, we demonstrate how these model membrane systems allow modulation of the PR photocycle by variation of the discoidal diameter (i.e., 10 or 12 nm), bilayer thickness (i.e., 23 or 26.5 Å), and degree of saturation of the lipid acyl chain. Nanodiscs also offer a highly stable environment of relevance to potential device applications

Crystal Structure of Recoverin with Calcium Ions Bound to Both Functional EF Hands

Recoverin (Rv), a small Ca²⁺-binding protein that inhibits rhodopsin kinase (RK), has four EF hands, two of which are functional (EF2 and EF3). Activation requires Ca²⁺ in both EF hands, but crystal structures have never been observed with Ca²⁺ ions in both sites; all previous structures have Ca²⁺ bound to only EF3. We suspected that this was due to an intermolecular crystal contact between T80 and a surface glutamate (E153) that precluded coordination of a Ca²⁺ ion in EF2. We constructed the E153A mutant, determined its X-ray crystal structure to 1.2 Å resolution, and showed that two Ca²⁺ ions are bound, one in EF3 and one in EF2. Additionally, several other residues are shown to adopt conformations in the 2Ca²⁺ structure not seen previously and not seen in a second structure of the E153A mutant containing Na⁺ instead of Ca²⁺ in the EF2 site. The side-chain rearrangements in these residues form a 28 Å allosteric cascade along the surface of the protein connecting the Ca²⁺-binding site of EF2 with the active-site pocket responsible for binding RK

Photochromic Bacteriorhodopsin Mutant with High Holographic Efficiency and Enhanced Stability via a Putative Self-Repair Mechanism

The <b>Q</b> photoproduct of bacteriorhodopsin (BR) is the basis of several biophotonic technologies that employ BR as the photoactive element. Several blue BR (bBR) mutants, generated by using directed evolution, were investigated with respect to the photochemical formation of the <b>Q</b> state. We report here a new bBR mutant, D85E/D96Q, which is capable of efficiently converting the entire sample to and from the <b>Q</b> photoproduct. At pH 8.5, where <b>Q</b> formation is optimal, the <b>Q</b> photoproduct requires 65 kJ mol^‑1 of amber light irradiation (590 nm) for formation and 5 kJ mol^‑1 of blue light (450 nm) for reversion, respectively. The melting temperature of the resting state and <b>Q</b> photoproduct, measured via differential scanning calorimetry, is observed at 100 °C and 89 °C at pH 8.5 or 91 °C and 82 °C at pH 9.5, respectively. We hypothesize that the protein stability of D85E/D96Q compared to other blue mutants is associated with a rapid equilibrium between the blue form E85­(H) and the purple form E85(−) of the protein, the latter providing enhanced structural stability. Additionally, the protein is shown to be stable and functional when suspended in an acrylamide matrix at alkaline pH. Real-time photoconversion to and from the <b>Q</b> state is also demonstrated with the immobilized protein. Finally, the holographic efficiency of an ideal thin film using the <b>Q</b> state of D85E/D96Q is calculated to be 16.7%, which is significantly better than that provided by native BR (6–8%) and presents the highest efficiency of any BR mutant to date

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<i>meso</i>-Arylporpholactones and their Reduction Products

The rational syntheses of <i>meso</i>-tetraaryl-3-oxo-2-oxaporphyrins <b>5</b>, known as porpholactones, via MnO₄^–-mediated oxidations of the corresponding <i>meso</i>-tetraaryl-2,3-dihydroxychlorins (<b>7</b>) is detailed. Since chlorin <b>7</b> is prepared from the parent porphyrin <b>1</b>, this amounts to a 2-step replacement of a pyrrole moiety in <b>1</b> by an oxazolone moiety. The stepwise reduction of the porpholactone <b>5</b> results in the formation of chlorin analogues, <i>meso</i>-tetraaryl-3-hydroxy-2-oxachlorin (<b>11</b>) and <i>meso</i>-tetraaryl-2-oxachlorins (<b>12</b>). The reactivity of <b>11</b> with respect to nucleophilic substitution by O-, N-, and S-nucleophiles is described. The profound photophysical consequences of the formal replacement of a pyrrole with an oxazolone (porphyrin-like chromophore) or (substituted) oxazole moiety (chlorin-like chromophore with, for the parent oxazolochlorin <b>12</b>, red-shifted Q_<i>x</i> band with enhanced oscillator strengths) are detailed and rationalized on the basis of SAC–CI and MNDO-PSDCI molecular orbital theory calculations. The single crystal X-ray structures of the porpholactones point at a minor steric interaction between the carbonyl oxygen and the flanking phenyl group. The essentially planar structures of all chromophores in all oxidation states prove that the observed optical properties originate from the intrinsic electronic properties of the chromophores and are not subject to conformational modulation

<i>meso</i>-Arylporpholactones and their Reduction Products

The rational syntheses of <i>meso</i>-tetraaryl-3-oxo-2-oxaporphyrins <b>5</b>, known as porpholactones, via MnO₄^–-mediated oxidations of the corresponding <i>meso</i>-tetraaryl-2,3-dihydroxychlorins (<b>7</b>) is detailed. Since chlorin <b>7</b> is prepared from the parent porphyrin <b>1</b>, this amounts to a 2-step replacement of a pyrrole moiety in <b>1</b> by an oxazolone moiety. The stepwise reduction of the porpholactone <b>5</b> results in the formation of chlorin analogues, <i>meso</i>-tetraaryl-3-hydroxy-2-oxachlorin (<b>11</b>) and <i>meso</i>-tetraaryl-2-oxachlorins (<b>12</b>). The reactivity of <b>11</b> with respect to nucleophilic substitution by O-, N-, and S-nucleophiles is described. The profound photophysical consequences of the formal replacement of a pyrrole with an oxazolone (porphyrin-like chromophore) or (substituted) oxazole moiety (chlorin-like chromophore with, for the parent oxazolochlorin <b>12</b>, red-shifted Q_<i>x</i> band with enhanced oscillator strengths) are detailed and rationalized on the basis of SAC–CI and MNDO-PSDCI molecular orbital theory calculations. The single crystal X-ray structures of the porpholactones point at a minor steric interaction between the carbonyl oxygen and the flanking phenyl group. The essentially planar structures of all chromophores in all oxidation states prove that the observed optical properties originate from the intrinsic electronic properties of the chromophores and are not subject to conformational modulation

<i>meso</i>-Arylporpholactones and their Reduction Products

The rational syntheses of <i>meso</i>-tetraaryl-3-oxo-2-oxaporphyrins <b>5</b>, known as porpholactones, via MnO₄^–-mediated oxidations of the corresponding <i>meso</i>-tetraaryl-2,3-dihydroxychlorins (<b>7</b>) is detailed. Since chlorin <b>7</b> is prepared from the parent porphyrin <b>1</b>, this amounts to a 2-step replacement of a pyrrole moiety in <b>1</b> by an oxazolone moiety. The stepwise reduction of the porpholactone <b>5</b> results in the formation of chlorin analogues, <i>meso</i>-tetraaryl-3-hydroxy-2-oxachlorin (<b>11</b>) and <i>meso</i>-tetraaryl-2-oxachlorins (<b>12</b>). The reactivity of <b>11</b> with respect to nucleophilic substitution by O-, N-, and S-nucleophiles is described. The profound photophysical consequences of the formal replacement of a pyrrole with an oxazolone (porphyrin-like chromophore) or (substituted) oxazole moiety (chlorin-like chromophore with, for the parent oxazolochlorin <b>12</b>, red-shifted Q_<i>x</i> band with enhanced oscillator strengths) are detailed and rationalized on the basis of SAC–CI and MNDO-PSDCI molecular orbital theory calculations. The single crystal X-ray structures of the porpholactones point at a minor steric interaction between the carbonyl oxygen and the flanking phenyl group. The essentially planar structures of all chromophores in all oxidation states prove that the observed optical properties originate from the intrinsic electronic properties of the chromophores and are not subject to conformational modulation