Photoactive Protein Research Articles

Development of an automated fluorescence microscopy system for photomanipulation of genetically encoded photoactivatable proteins (optogenetics) in live cells

Photomanipulation of genetically encoded light-sensitive protein activity, also known as optogenetics, is one of the most innovative recent microscopy techniques in the fields of cell biology and neurobiology. Although photomanipulation is usually performed by diverting the photobleaching mode of a confocal laser microscope, photobleaching by the laser scanning unit is not always suitable for photoactivation. We have developed a simple automated wide-field fluorescence microscopy system for the photomanipulation of genetically encoded photoactivatable proteins in live cells. An electrically automated fluorescence microscope can be controlled through MetaMorph imaging software, making it possible to acquire time-lapse, multiwavelength images of live cells. Using the journal (macro recording) function of MetaMorph, we wrote a macro program to change the excitation filter for photoactivation and illumination area during the intervals of image acquisition. When this program was run on the wide-field fluorescence microscope, cells expressing genetically encoded photoactivatable Rac1, which is activated under blue light, showed morphological changes such as lamellipodial extension and cell surface ruffling in the illuminated region. Using software-based development, we successfully constructed a fully automated photoactivation microscopy system for a mercury lamp-based fluorescence microscope.

A Molecular Voltmeter Based on Fluorescence Dynamics

In this issue, Hou et al. (1) introduce an elegant method for measuring the absolute voltage across a cell membrane with an accuracy of 10 mV. Their study presents an incisive approach to voltage imaging in which the voltage value is encoded in the temporal dynamics of fluorescence rather than in the raw fluorescence intensity. Together with an engineered microbial rhodopsin with properties amenable to the approach, the method provides a genetically encoded optical read-out independent of the reporter’s expression level and insensitive to variations in common experimental parameters. Their work presents a conceptually new avenue toward developing probes for membrane voltage useful for myriad systems from bacteria to mammalian tissue.

Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-aequorin reporter

Calcium (Ca2+) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca2+ signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca2+ signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca2+ signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca2+ imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca2+ sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca2+-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca2+ signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca2+ dynamics in intact plants. We describe a simple method to image Ca2+ signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca2+ signals. It is shown that Ca2+ signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.

Light-Harvesting Lipid Vesicles Incorporated with Proteorhodopsins and Photosystem II; Generation of Photo-Induced Proton Gradients and Extended Absorbing Light Spectrum

The most common uses of electrochemical potentials in living cells are to use for energy storages, which are transferable to various essential cellular activities, i.e. the generation of ATP or NADPH, and ions or molecular transport across a membranes. Often, the electrochemical potentials (i.e. proton gradient) are produced by activation of light-harvesting membrane proteins. Depending on the cells from various species, different membrane protein complexes are acting similar roles, in terms of generating an electrochemical potential, by absorbing different light-spectrum. For example, proteorhodopsin and photosystemII are well-known photoactive transmembrane proteins, functioning as a light-driven proton pump in marine bacteria or eukaryotes, and a light-driven proton production in plants, respectively. In their natural habitats, not only their biological roles, but also their activating absorption wavelengths are very different; PR functions as a light-driven H+ pump activated by green light, while as PS II captures photons of light to energize electrons, which replaced by oxidizing water to form hydrogen ions by absorbing blue and red light. Although their light-driven mechanisms and their utilizing wavelength bands are very much different, both systems can result a similar proton gradient across the membrane. Consequently, we hypothesized that we can build an artificial light-harvesting system, which allows utilizing multiple maximum absorption bands. By reconstitution of two different purified proteins into a single giant vesicle, we could successfully build the hybrid light-harvesting liposomes incorporated both with PR and PSII, activated by the wide wavelength bands, from blue up to NIR. This new system, generating a improved gradient of electrochemical potential across a membrane, can be used as numerous energy storages in vitro, providing photo-induced energies for flagella rotation, NADPH synthesis or ATP synthesis with highly improved light efficiencies.

Utilising Super-Resolution Palm Imaging in Fission Yeast

Super-resolution techniques such as PALM and STORM are an exciting development allowing researchers to break the diffraction limit imposed by the diffraction of light. Our group works on understanding the systems of DNA repair and replication restart using the model organism Schizosaccharomyces pombe (fission yeast). A recent paper by Lando et al., utilised PALM super-resolution imaging to count the number of a protein tagged with the photo-activatable protein mEos2 in fission yeast. The potential to determine the precise stoichiometry of protein complexes in vivo would be an important breakthrough. To do this it is necessary to establish the ideal protocols for super-resolution imaging in S. pombe, and what the limitations of this technique are. We present the results of work with Cdc22, a protein highly expressed throughout the cell in fission yeast, known to form hexamers, with a control mutant impaired in hexamer formation. The protein was labelled genomically with the photo-activatable protein mEos2 to allow it to be visualised. We have established optimal excitation power and temporal regimes to visualise Cdc22 in S. pombe, as well as protocols to significantly reduce intracellular background fluorescence.

Remote Control of Diverse Cytoskeletal Motors using Light-Activated Gearshifting

Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Protein engineering has been used to modify cytoskeletal motors for dynamic control of activity and directionality, providing direct tests of structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. We have previously1 created myosin motors that can be signaled to switch directions in response to changes in [Ca2+]. Light is a more versatile control signal2 because it can be precisely modulated in space and time, and is generally orthogonal to cellular signaling. Here we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears - speed up, slow down, or switch directions - when exposed to blue light. Our structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. We have used in vitro motility assays to confirm robust spatiotemporal control over motor function and to characterize the kinetics of optical gearshifting. Our modular approach has yielded controllable motors for both actin-based and microtubule-based transport. Genetically encoded light-responsive motors will expand the optogenetics toolkit, complementing precise perturbations of ion channels and intracellular signaling with spatiotemporal control of cytoskeletal transport and contractility.1. Chen, L., Nakamura, M., Schindler, T.D., Parker, D. & Bryant, Z. Engineering controllable bidirectional molecular motors based on myosin. Nature nanotechnology 7, 252-6 (2012).2. Walter, W.J. & Diez, S. Myosin shifts into reverse gear. Nature nanotechnology7, 213-4 (2012).

Sub-diffraction imaging with confocal fluorescence microscopy by stochastic photobleaching

We propose a single molecule localization method which takes advantage of stochastic photobleaching to improve the resolution of confocal fluorescence microscopy. By detecting the stochastic intensity loss of fluorophores, each fluorophore in the field can be localized. When all locations are known, a sub-diffraction image can be retrieved through single molecule localization algorithms. A confocal scheme is used to record the bleaching process of the sample. Each fluorophore can be localized from the recorded streaming followed by image subtraction. Compared with other single molecule localization concepts such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM), this method does not require a laser cycling equipment and the pixel size is no longer limited by the size of CCD. This technique works well with common fluorescent dyes and does not require the use of engineered photoactivatable proteins or photoswitchable synthetic dye pairs.

Photoreceptors for a light biotransducer: a comparative study of the electrical responses of two (type-1) opsins

The increasing interest in photoactivated proteins as natural replacements for standard inorganic materials in photocells leads to the comparison analysis of bacteriorhodopsin and proteorhodopsin, two widely diffused proteins belonging to the family of type-1 opsins. These proteins share similar behaviors but exhibit relevant differences in the sequential chain of the amino acids constituting their tertiary structure. The use of an impedance network analog to model the protein main features provides a microscopic interpretation of a set of experiments on their photo-conductance properties. In particular, this model links the protein electrical responses to the tertiary structure and to the interactions between neighboring amino acids. The same model is also used to predict the small-signal response in terms of the Nyquist plot. Interestingly, these rhodopsins are found to behave like a wide-gap semiconductor with intrinsic conductivities of the order of 10−7 S cm−1.

Isolation of a new Pseudomonas halophila strain possess bacteriorhodopsin-like protein by a novel method for screening of photoactive protein producing bacteria

Bacteriorhodopsin (bR) is a transmembrane protein deposited in the purple membrane of Halobacterium salinarum which absorbs energy from photons to create a photo-induced proton gradient across the membrane. A bR molecule can be considered as a natural solar device transforming light into other types of energy and therefore is of interest for a wide range of applications including two and three-dimensional memory storage, optical data processing, artificial cells, holographic media, the artificial retina and photo sensor devices. H. salinarum is a slow-growing, halophilic Archaea present in red salt waters. The present study introduces a novel bR-like pigment from a new strain of Pseudomonas halophila (with registered accession number KC959570 in the NCBI databank) which has a very significant degree of light-dependent activity. This is the first report on the presence of functional bR-like protein in the Pseudomonas family. The isolate is a fast-growing, halophilic bacterium and is comparable with other photoactive protein producer microorganisms. Also, in the present study a novel isolation method for screen light-stimulating protein producing microorganisms is introduced. For this purpose 2,3,5-triphenyltetrazolium chloride (TTC) was employed for the first time as an artificial hydrogen acceptor in the proton-transfer processes. The TTC test is an easy and susceptible method for estimating hydrogen production during the proton transport process. This is the first report of the use of TTC for photo activity measurement and selection of bacteria containing light dependent proteins.

Directed evolution of bacteriorhodopsin for applications in bioelectronics

In nature, biological systems gradually evolve through complex, algorithmic processes involving mutation and differential selection. Evolution has optimized biological macromolecules for a variety of functions to provide a comparative advantage. However, nature does not optimize molecules for use in human-made devices, as it would gain no survival advantage in such cooperation. Recent advancements in genetic engineering, most notably directed evolution, have allowed for the stepwise manipulation of the properties of living organisms, promoting the expansion of protein-based devices in nanotechnology. In this review, we highlight the use of directed evolution to optimize photoactive proteins, with an emphasis on bacteriorhodopsin (BR), for device applications. BR, a highly stable light-activated proton pump, has shown great promise in three-dimensional optical memories, real-time holographic processors and artificial retinas.

The Orange Carotenoid Protein: a blue-green light photoactive protein

This review focuses on the Orange Carotenoid Protein (OCP) which is the first photoactive protein identified containing a carotenoid as the photoresponsive chromophore. This protein is essential for the triggering of a photoprotective mechanism in cyanobacteria which decreases the excess absorbed energy arriving at the photosynthetic reaction centers by increasing thermal dissipation at the level of the phycobilisomes, the cyanobacterial antenna. Blue-green light causes structural changes within the carotenoid and the protein, converting the orange inactive form into a red active form. The activated red form interacts with the phycobilisome and induces the decrease of phycobilisome fluorescence emission and of the energy arriving to the photosynthetic reaction centers. The OCP is the light sensor, the signal propagator and the energy quencher. A second protein, the Fluorescence Recovery Protein (FRP), is needed to detach the red OCP from the phycobilisome and its reversion to the inactive orange form. In the last decade, in vivo and in vitro mechanistic studies combined with structural and genomic data resulted in both the discovery and a detailed picture of the function of the OCP and OCP-mediated photoprotection. Recent structural and functional results are emphasized and important previous results will be reviewed. Similarities to other blue-light responsive proteins will be discussed.

Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile

So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.

Spectral phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR

The phasor global analysis algorithm is common for fluorescence lifetime applications, but has only been recently proposed for spectral analysis. Here the phasor representation and fingerprinting is exploited in its second harmonic to determine the number and spectra of photo-activated states as well as their conversion dynamics. We follow the sequence of photo-activation of proteins over time by rapidly collecting multiple spectral images. The phasor representation of the cumulative images provides easy identification of the spectral signatures of each photo-activatable protein.

Characterization of a Xanthorhodopsin-homologue from the North Pole

Rhodopsins belong to a family of membrane-embedded photoactive retinylidene proteins. One opsin gene was isolated from <TEX>${\beta}$</TEX>-proteobacterium (IMCC9480) which had been collected at the North Pole. It is very similar to Xanthorhodopin (XR) of HTCC2181. In this study, we carried out basic characterization of the rhodopsin. It has <TEX>${\lambda}max$</TEX> of 536, 554, and 546 nm at pH 4.0, 7.0, and 10.0, respectively. Since the pKa of its proton acceptor is around 6.27, we measured its proton pumping activity and photocycling rate at pH 8.0. It has a typical proton acceptor (D99) and donor (E110) which mediate proton translocation from intracellular to extracellular region when deduced from the sequence alignments. On the basis of in vitro proton pumping activity, it was proposed to have fast photocycling rate with M and O intermediates, indicating that it is a typical ion-pumping rhodopsin. Since the XR has not yet been expressed in any other heterologous expression system, we tried to get much more information about the XR through the XR-homologue rhodopsin.

Crystal structure of the FRP and identification of the active site for modulation of OCP-mediated photoprotection in cyanobacteria

Photosynthetic reaction centers are sensitive to high light conditions, which can cause damage because of the formation of reactive oxygen species. To prevent high-light induced damage, cyanobacteria have developed photoprotective mechanisms. One involves a photoactive carotenoid protein that decreases the transfer of excess energy to the reaction centers. This protein, the orange carotenoid protein (OCP), is present in most cyanobacterial strains; it is activated by high light conditions and able to dissipate excess energy at the site of the light-harvesting antennae, the phycobilisomes. Restoration of normal antenna capacity involves the fluorescence recovery protein (FRP). The FRP acts to dissociate the OCP from the phycobilisomes by accelerating the conversion of the active red OCP to the inactive orange form. We have determined the 3D crystal structure of the FRP at 2.5 Å resolution. Remarkably, the FRP is found in two very different conformational and oligomeric states in the same crystal. Based on amino acid conservation analysis, activity assays of FRP mutants, FRP:OCP docking simulations, and coimmunoprecipitation experiments, we conclude that the dimer is the active form. The second form, a tetramer, may be an inactive form of FRP. In addition, we have identified a surface patch of highly conserved residues and shown that those residues are essential to FRP activity.

Application of Nanoparticle Antioxidants to Enable Hyperstable Chloroplasts for Solar Energy Harvesting

AbstractThe chloroplast contains densely stacked arrays of light‐harvesting proteins that harness solar energy with theoretical maximum glucose conversion efficiencies approaching 12%. Few studies have explored isolated chloroplasts as a renewable, abundant, and low cost source for solar energy harvesting. One impediment is that photoactive proteins within the chloroplast become photodamaged due to reactive oxygen species (ROS) generation. In vivo, chloroplasts reduce photodegradation by applying a self‐repair cycle that dynamically replaces photodamaged components; outside the cell, ROS‐induced photodegradation contributes to limited chloroplast stability. The incorporation of chloroplasts into synthetic, light‐harvesting devices will require regenerative ROS scavenging mechanisms to prolong photoactivity. Herein, we study ROS generation within isolated chloroplasts extracted from Spinacia oleracea directly interfaced with nanoparticle antioxidants, including dextran‐wrapped nanoceria (dNC) previously demonstrated as a potent ROS scavenger. We quantitatively examine the effect of dNC, along with cerium ions, fullerenol, and DNA‐wrapped single‐walled carbon nanotubes (SWCNTs), on the ROS generation of isolated chloroplasts using the oxidative dyes, 2’,7’‐ dichlorodihydrofluorescein diacetate (H2DCF‐DA) and 2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide sodium salt (XTT). Electrochemical measurements confirm that chloroplasts processed from free solution can generate power under illumination. We find dNC to be the most effective of these agents for decreasing oxidizing species and superoxide concentrations whilst preserving chloroplast photoactivity at concentrations below 5 μM, offering a promising mechanism for maintaining regenerative chloroplast photoactivity for light‐harvesting applications.

Guiding lights: recent developments in optogenetic control of biochemical signals

Optogenetics arises from the innovative application of microbial opsins in mammalian neurons and has since been a powerful technology that fuels the advance of our knowledge in neuroscience. In recent years, there has been growing interest in designing optogenetic tools extendable to broader cell types and biochemical signals. To date, a variety of photoactivatable proteins (refers to induction of protein activity in contrast to fluorescence) have been developed based on the understanding of plant and microbial photoreceptors including phototropins, blue light sensors using flavin adenine dinucleotide proteins, cryptochromes, and phytochromes. Such tools offered researchers reversible, quantitative, and precise spatiotemporal control of enzymatic activity, protein-protein interaction, protein translocation, as well as gene transcription in cells and in whole animals. In this review, we will briefly introduce these photosensory proteins, describe recent developments in optogenetics, and compare and contrast different methods based on their advantages and limitations.

Volume-conserving trans–cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography

Trans-to-cis isomerization, the key reaction in photoactive proteins, cannot usually occur through the standard one-bond-flip mechanism. Due to spatial constraints imposed by a protein environment, isomerization is likely to proceed via a “volume-conserving” mechanism in which highly-choreographed atomic motions are expected, the details of which have not yet been directly observed. Here we employ time-resolved X-ray crystallography to structurally visualize isomerization of the p-coumaric acid chromophore in photoactive yellow protein with 100 picosecond time resolution and 1.6 Å spatial resolution. The structure of the earliest intermediate (IT) resembles a highly-strained transition state in which the torsion angle is located halfway between the trans and cis isomers. The reaction trajectory of IT bifurcates into two structurally distinct cis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue via E46Q mutation, which switches off the bicycle-pedal pathway.

Determination of two-photon photoactivation rates of fluorescent proteins

The application of two-photon activation of photoactivatable fluorescent proteins is limited by a lack of information about two-photon activation rates. Here we present rates for the commonly used photoactivatable proteins PAmCherry, PAmKate and PA-GFP at different wavelengths using a novel method that allows us to determine the two-photon activation rates directly, independent of any reference data, with microscopic sample volumes. We show that PAmCherry features the highest rates of the tested proteins at 700 nm activation wavelength followed by PAmKate. Towards longer wavelengths, two-photon activation rates decrease for all three proteins. For PAmCherry, our data contradicts an activation model relying solely on two-photon activation and suggests additional activation pathways requiring at least two absorption steps. Our method is readily expandable to other photoactivatable fluorescent molecules. The presented results allow optimization of experimental conditions in spectroscopic and imaging techniques such as super-resolution fluorescence microscopy.

Orange Carotenoid Protein; Characterization with Resonance Raman Spectroscopy

In photosynthetic organisms, high light intensities can result in cellular damage from the production of dangerous oxygen species. Cyanobacteria, also known as blue-green bacteria, blue-green algae, and Cyanophyta, is a phylum of bacteria that obtain their energy through photosynthesis. Reactive oxygen species (ROS) are created from the over-reduction of photosynthetic components, like the plastoquinone pool. Orange carotenoid protein (OCP) acts as a protective mechanism in certain cyanobacteria as it shuttles energy away from the light harvesting apparatuses (in cyanobacteria they are usually phycobilisomes). For my PhD, I am biophysically characterizing this protein with Raman spectroscopy and other techniques. OCP is a photoactive protein that changes confirmation when exposed to blue light; it contains a carotenoid ligand, 3'-hydroxyechinenone. I have purified the his-tagged OCP (from the bacteria Synechocystis PCC 6803) with a nickel column, and am currently using Raman spectroscopy to study the protein's inner carotenoid to deduce information about the vibrational sublevels, and thus the protein environment surrounding the carotenoid. My experiments consist of comparison between the orange (inactive) and red (active- after light exposure) structural forms of the protein. I will soon be using Raman spectroscopy on mutants of the protein as well. Other techniques that I will use for characterization include FTIR (fourier transform infrared), two photon absorption spectroscopy and analytical ultracentrifugation (AUC).