Retinal Binding to Apo-Gloeobacter Rhodopsin: The Role of pH and Retinal–Carotenoid Interaction

Over the past few decades, the structure, functions, properties, and molecular mechanisms of retinal proteins have been studied extensively. The newly studied retinal protein Gloeobacter rhodopsin (gR) acts as a light-driven proton pump, transferring a proton from the cytoplasmic region to the extracellular region of a cell following light absorption. It was previously shown that gR can bind the carotenoid salinixanthin (sal). In the present study, we report the effect of pH on the binding of retinal to the apo-protein of gR, in the presence and absence of sal, to form the gR pigment. We found that binding at different pH levels reflects the titration of two different protein residues, one at the lower p<i>K</i>_a 3.5 and another at the higher p<i>K</i>_a 8.4, that affect the pigment’s formation. The maximum amount of pigment was formed at pH 5, both with and without the presence of sal. The introduction of sal accelerates the rate of pigment formation by a factor of 190. Furthermore, it is suggested that occupation of the binding site by the retinal chromophore induces protein conformational alterations which in turn affect the carotenoid conformation, which precedes the formation of the retinal–protein covalent bond. Our examination of synthetic retinal analogues in which the ring structure was modified revealed that, in the absence of sal, the retinal ring structure affects the rate of pigment formation and that the intact structure is needed for efficient pigment formation. However, the presence of sal abolishes this effect, and all-trans retinal and its modified ring analogues bind at a similar rate

Absorption maximum and pK_a value of wild-type and various GR mutants.

<p>Absorption maximum and pK_a value of wild-type and various GR mutants.</p

DNA binding activity of <i>Anabaena</i> sensory rhodopsin transducer probed by fluorescence correlation spectroscopy

<div><p><i>Anabaena</i> sensory rhodopsin transducer (ASRT) is believed to be a major player in the photo-signal transduction cascade, which is triggered by <i>Anabaena</i> sensory rhodopsin. Here, we characterized DNA binding activity of ASRT probed by using fluorescence correlation spectroscopy. We observed clear decrease of diffusion coefficient of DNA upon binding of ASRT. The dissociation constant, K_D, of ASRT to 20 bp-long DNA fragments lied in micro-molar range and varied moderately with DNA sequence. Our results suggest that ASRT may interact with several different regions of DNA with different binding affinity for global regulation of several genes that need to be activated depending on the light illumination.</p></div

Schematic model of energy production in <i>Gloeobacter</i>.

<p>The photosynthetic apparatus of <i>Gloeobacter</i> is reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110643#pone.0110643-Koenig1" target="_blank">[51]</a>.</p

Pylogenetic tree and alignment.

<p>(a) Phylogenetic tree of 22 microbial opsin sequences, representing phylogenetic relationship between <i>Gloeobacter</i> rhodopsin and related proteins from archaea to algae. Analysis was conducted by ClustalW. A dotted line on a phenogram indicates a negative branch length, a common result of averaging by the DNA STAR software. GR: <i>Gloeobacter</i> rhodopsin (accession number; NP_923144). Red23 and REDr6a5a2 were collected from Red Sea. GRP/BPR: green/blue-absorbing PRs. PalE6 was collected from Antarctic ocean. RhodospR: rhodopsin from <i>Rhodospirillales sp</i>. XR: Xanthorhodopsin from <i>Salinibacter ruber</i>. FulviR: rhodopsin from <i>Fulvimarina</i>. ArchaerR1: Archaerhodopsin 1. PYR: rhodopsin from <i>Pyrocystis lunula</i>. BR: <i>Halobacterium salinarum</i> bacteriorhodopsin. RoseiR: rhodopsin from <i>Roseiflexus sp</i>.MarinoR: rhodopsin from <i>Marinobacter</i>. MethyloR: rhodopsin from <i>Methylophilales</i>.LR: rhodopsin from <i>Leptosphaeria maculans</i>. NR: rhodopsin from <i>Neurospora crassa</i>. AR: rhodopsin from <i>Acetabularia acetabulum</i>. SRI: <i>H. salinarum</i> sensory rhodopsin I. NpSRII: <i>Natronomonas pharaonis</i> sensory rhodopsin II. ASR: sensory rhodopsin from <i>Anabaena (Nostoc) sp.</i>CSOA: <i>Chlamydomonas reinhardtii</i> sensory rhodopsin A. (b) Alignment of the sequences of GR, XR, PR, RoseiR, MarinoR, MethyloR and BR. GR contains the functionally important residues for proton transport, including homologues of Arg82, Asp85, Trp86, Asp96, Trp182, Tyr185, Asp212, and Lys216, with some of the numbering and helical segments (marked by arrows) for BR. There are 22 residues (marked with black boxes) common to all seven proteins (in one case, Asp/Glu substitutions). The Asp121 (Asp85 in BR) should be the proton acceptor of the retinal Schiff base. The Glu132 (Asp96 in BR) may be the proton donor.</p

Action spectra for light-driven proton translocation in <i>Gloeobacter</i> cells (solid line).

<p>Sphaeroplasts of <i>Gloeobacter</i> cells were illuminated through the filters transmitting at 700, 650, 600, 550, 500, 450, and 400 nm. The efficiency of the sphaeroplasts (after the photosynthesis inhibition by DCMU treatment (5×10⁻⁵mol l⁻¹)) was most efficient at 550 nm (dashed line). Integrated H⁺ pumping amount is presented by blank column. Concentration of synthesized ATP is presented by slashed column.</p

Absorption spectra and titration curves of GR and its mutants.

<p>Insets show photos of purified WT and mutant GR in 50 mM Tris (pH 7), 150 mM NaCl, and 0.02% DDM. The pH titration curves indicate the pH dependence of the absorption maxima of broad spectral range. The pK_a values are shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110643#pone-0110643-t001" target="_blank">table 1</a>.</p

Immunoblot and spectroscopy analysis.

<p>(a) Immunoblot analysis of the <i>Gloeobacter</i> cells and the His-tagged GR protein. The immunoblot used anti-GR antibody for the membrane protein fraction of <i>Gloeobacter</i> cells and anti-His-tag antibody for the protein expressed in <i>E. coli</i>. The whole cells (C) were sonicated and lysates were ultracentrifuged. The lysates were separated into supernatant (A: soluble protein) and pellet (B: membrane protein). D: purified GR from <i>E. coli</i>. (b) Absorption spectra of intact cells and membranes of <i>G. violaceus</i>. The spectrum of <i>G. violaceus</i> is shown with solid line. Chl a (chlorophyll a), Car (carotenoid), PE (phycoerythrin), GR, and PC (phycocyanin) are marked by the arrows. The spectrum of <i>G</i>. <i>violaceus</i> membrane is shown by the dashed line. The spectrum of purified GR in 0.02% DDM, isolated from <i>E. coli</i> is shown by dotted line.</p

pH Dependence of Anabaena Sensory Rhodopsin: Retinal Isomer Composition, Rate of Dark Adaptation, and Photochemistry

Microbial rhodopsins are photoactive proteins, and their binding site can accommodate either all-trans or 13-cis retinal chromophore. The pH dependence of isomeric composition, dark-adaptation rate, and primary events of Anabaena sensory rhodopsin (ASR), a microbial rhodopsin discovered a decade ago, are presented. The main findings are: (a) Two p<i>K</i>_a values of 6.5 and 4.0 assigned to two different protein residues are observed using spectroscopic titration experiments for both ground-state retinal isomers: all-trans, 15-anti (AT) and 13-cis, 15-syn (13C). The protonation states of these protein residues affect the absorption spectrum of the pigment and most probably the isomerization process of the retinal chromophore. An additional p<i>K</i>_a value of 8.5 is observed only for 13C-ASR. (b) The isomeric composition of ASR is determined over a wide pH range and found to be almost pH-independent in the dark (>96% AT isomer) but highly pH-dependent in the light-adapted form. (c) The kinetics of dark adaptation is recorded over a wide pH range, showing that the thermal isomerization from 13C to AT retinal occurs much faster at high pH rather than under acidic conditions. (d) Primary photochemical events of ASR at pH 5 are recorded using VIS hyperspectral pump–probe spectroscopy with <100 fs resolution and compared with the previously recorded results at pH 7.5. For AT-ASR, these are shown to be almost pH-independent. However, photochemistry of 13C-ASR is pH-dependent and slowed down in acidic environments

Protein–protein docking to predict the 3D protein structure of AbHeR and AbGS.

AbHeR and AbGS are indicated as transparent cyan and red helices, respectively. The distances between the hydrogen bonds of interacting amino acids of AbHeR and AbGS were calculated by polar interaction tool in PyMOL and indicated using a yellow dotted line. The key active sites of AbGS are present in the 2 AbGS monomers of dodecamer, and the positions of the key active sites of the 2 different monomers in AbGS are indicated in yellow and white text. The positions of amino acids in AbHeR are indicated in blue text. (A, D, and E) Docking parts of key active sites in AbGS and AbHeR. (B and C) GS 3D structure (PDB: 6su3.1.A) in a position with bound Glu. (D and E) The positions of the docking prediction are indicated with white dotted circles. (B and D) Above and (C and E) top views of the positions are shown. (TIF)</p