Early-stage dynamics of chloride ion-pumping rhodopsin revealed by a femtosecond X-ray laser.

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.

A simple protocol for the isolation of proteorhodopsins of the dinoflagellate Oxyrrhis marina.

For the first time, native proteorhodopsins of the marine dinoflagellate Oxyrrhis marina were isolated. Total cell membrane fractions were minced in a bead beater and solubilized with the detergent Triton X-100. Subsequent sucrose density gradient centrifugation resulted in three or four red-colored bands. Nonsolubilized, but still red colored, membranes sedimented at the bottom. For each of these bands, absorbance maxima were registered at approximately 514-516 nm with shoulders toward shorter wavelengths (470-490 nm). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the uppermost band represented free retinal chromophore, as it contained no protein. The other bands were almost pure proteorhodopsin fractions as the banding patterns showed one major protein of 25 kDa. Tryptic, in-gel digestion of the 25 kDa proteins and of faint protein bands above and below 25 kDa was followed by mass spectrometry, confirming these protein bands to consist, nearly exclusively, proteorhodopsins. Only single peptides of few other proteins were detected. In total, at least seven predicted proteorhodopsin protein sequences were experimentally verified.

From Gene to Function: Cell-Free Electrophysiological and Optical Analysis of Ion Pumps in Nanodiscs.

Nanodiscs that hold a lipid bilayer surrounded by a boundary of scaffold proteins have emerged as a powerful tool for membrane protein solubilization and analysis. By combining nanodiscs and cell-free expression technologies, even completely detergent-free membrane protein characterization protocols can be designed. Nanodiscs are compatible with various techniques, and due to their bilayer environment and increased stability, they are often superior to detergent micelles or liposomes for membrane protein solubilization. However, transport assays in nanodiscs have not been conducted so far, due to limitations of the two-dimensional nature of nanodisc membranes that offers no compartmentalization. Here, we study Krokinobacter eikastus rhodopsin-2 (KR2), a microbial light-driven sodium or proton pump, with noncovalent mass-spectrometric, electrophysiological, and flash photolysis measurements after its cotranslational insertion into nanodiscs. We demonstrate the feasibility of adsorbing nanodiscs containing KR2 to an artificial bilayer. This allows us to record light-induced capacitive currents that reflect KR2's ion transport activity. The solid-supported membrane assay with nanodisc samples provides reliable control over the ionic condition and information of the relative ion activity of this promiscuous pump. Our strategy is complemented with flash photolysis data, where the lifetimes of different photointermediates were determined at different ionic conditions. The advantage of using identical samples to three complementary approaches allows for a comprehensive comparability. The cell-free synthesis in combination with nanodiscs provides a defined hydrophobic lipid environment minimizing the detergent dependence often seen in assays with membrane proteins. KR2 is a promising tool for optogenetics, thus directed engineering to modify ion selectivity can be highly beneficial. Our approach, using the fast generation of functional ion pumps incorporated into nanodiscs and their subsequent analysis by several biophysical techniques, can serve as a versatile screening and engineering platform. This may open new avenues for the study of ion pumps and similar electrogenic targets.

Biochemical Analysis of Microbial Rhodopsins.

Ion-pumping rhodopsins transfer ions across the microbial cell membrane in a light-dependent fashion. As the rate of biochemical characterization of microbial rhodopsins begins to catch up to the rate of microbial rhodopsin identification in environmental and genomic sequence data sets, in vitro analysis of their light-absorbing properties and in vivo analysis of ion pumping will remain critical to characterizing these proteins. As we learn more about the variety of physiological roles performed by microbial rhodopsins in different cell types and environments, observing the localization patterns of the rhodopsins and/or quantifying the number of rhodopsin-bearing cells in natural environments will become more important. Here, we provide protocols for purification of rhodopsin-containing membranes, detection of ion pumping, and observation of functional rhodopsins in laboratory and environmental samples using total internal reflection fluorescence microscopy. © 2016 by John Wiley & Sons, Inc.

Microbial rhodopsins of Halorubrum species isolated from Ejinoor salt lake in Inner Mongolia of China.

Microbial rhodopsins are photoactive proteins that use a retinal molecule as the photoactive center. Because of structural simplicity and functional diversity, microbial rhodopsins have been an excellent model system for structural biology. In this study, a halophilic archaea that has three microbial rhodopsin-type genes in its genome was isolated from Ejinoor salt lake in Inner Mongolia of China. A sequence of 16S rRNA showed that the strain belongs to Halorubrum genus and named Halorubrum sp. ejinoor (He). The translated amino acid sequences of its microbial rhodopsin-type genes suggest that they are homologs of archaerhodopsin (HeAR), halorhodopsin (HeHR) and sensory rhodopsin II (HeSRII). The mRNAs of three types of genes were detected by RT-PCR and their amounts were investigated by Real-Time RT-PCR. The amount of mRNA of HeSRII was the smallest and the amounts of of HeAR and HeHR were 30 times and 10 times greater than that of HeSRII. The results of light-induced pH changes suggested the presence of a light-driven proton pump and a light-driven chloride ion pump in the membrane vesicles of He. Flash induced absorbance changes of the He membrane fraction indicated that HeAR and HeHR are photoactive and undergo their own photocycles. This study revealed that three microbial rhodopsin-type genes are all expressed in the strain and at least two of them, HeAR and HeHR, are photochemically and physiologically active like BR and HR of Halobacterium salinarum, respectively. To our knowledge, this is the first report of physiological activity of HR-homolog of Halorubrum species.

Isolation of proteorhodopsin-bearing bacterium JL-3 from fresh water and characterization of the proteorhodopsin.

Proteorhodopsins (PRs), light-driven proton pumps, constitute the largest family of the microbial rhodopsins. PRs are widely distributed in the oceanic environment and freshwater, but no bacteria with PRs have been isolated from freshwater so far. To facilitate isolation of the bacteria with PR genes, we constructed a vector system that can be used to clone potential PR genes and render color changes when overexpressed in Escherichia coli. Using this method, we successfully isolated a strain with PR gene from freshwater and identified it as Exiguobacterium sp. JL-3. The full length PR gene was then cloned using the SEFA PCR method. Protein sequence alignment showed that JL-3_PR shares high sequence identity (84-89%) with the PRs from Exiguobacterium strains, but low sequence identity (< 38%) with other PRs. Surprisingly, we could not detect any proton-pumping activity in the native JL-3 cells and protoplasts, but the recombinant JL-3_PR do pump protons when overexpressed in E. coli. Sequence analysis further revealed that the PRs from Exiguobacterium had an unusual lysine as the proton donor instead of the typical acidic residue. These data suggest that JL-3_PR is a sensory PR rather than a proton pump.

The retinal structure of channelrhodopsin-2 assessed by resonance Raman spectroscopy.

Channelrhodopsin-2 mediates phototaxis in green algae by acting as a light-gated cation channel. As a result of this property, it is used as a novel optogenetic tool in neurophysiological applications. Structural information is still scant and we present here the first resonance Raman spectra of channelrhodopsin-2. Spectra of detergent solubilized and lipid-reconstituted protein were recorded under pre-resonant conditions to exclusively probe retinal in its electronic ground state. All-trans retinal was identified to be the favoured configuration of the chromophore but significant contributions of 13-cis were detected. Pre-illumination hardly changed the isomeric composition but small amounts of presumably 9-cis retinal were found in the light-adapted state. Spectral analysis suggested that the Schiff base proton is strongly hydrogen-bonded to a nearby water molecule.

Crystallization, X-ray diffraction analysis and SIRAS/molecular-replacenent phasing of three crystal forms of Anabaena sensory rhodopsin transducer.

Anabaena sensory rhodopsin transducer (ASRT) is a 14.7 kDa soluble signaling protein associated with the membrane-embedded light receptor Anabaena sensory rhodopsin (ASR) from Anabaena sp., a freshwater cyanobacterium. Crystals of ASRT were obtained in three different space groups, P4, C2 and P2(1)2(1)2(1), which diffract to 1.8, 2.1 and 2.0 angstroms, respectively. Phases for one of these crystal forms (P4) were obtained by SIRAS phasing using an iodide quick-soak derivative and a partial model was built. Phases for the remaining crystal forms were obtained by molecular replacement using the partial model from the P4 crystal form.

Xanthorhodopsin: a proton pump with a light-harvesting carotenoid antenna.

Energy transfer from light-harvesting carotenoids to chlorophyll is common in photosynthesis, but such antenna pigments have not been observed in retinal-based ion pumps and photoreceptors. Here we describe xanthorhodopsin, a proton-pumping retinal protein/carotenoid complex in the eubacterium Salinibacter ruber. The wavelength dependence of the rate of pumping and difference absorption spectra measured under a variety of conditions indicate that this protein contains two chromophores, retinal and the carotenoid salinixanthin, in a molar ratio of about 1:1. The two chromophores interact strongly, and light energy absorbed by the carotenoid is transferred to the retinal with a quantum efficiency of approximately 40%. The antenna carotenoid extends the wavelength range of the collection of light for uphill transmembrane proton transport.