Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein–protein interaction.

The second messengers, cyclic adenosine 3′-5′-monophosphate (cAMP) and cyclic guanosine 3′-5′-monophosphate (cGMP), play important roles in many animal cells by regulating intracellular signaling pathways and modulating cell physiology. Environmental cues like temperature, light, and chemical compounds can stimulate cell surface receptors and trigger the generation of second messengers and the following regulations. The spread of cAMP and cGMP is further shaped by cyclic nucleotide phosphodiesterases (PDEs) for orchestration of intracellular microdomain signaling. However, localized intracellular cAMP and cGMP signaling requires further investigation. Optogenetic manipulation of cAMP and cGMP offers new opportunities for spatio-temporally precise study of their signaling mechanism. Light-gated nucleotide cyclases are well developed and applied for cAMP/cGMP manipulation. Recently discovered rhodopsin phosphodiesterase genes from protists established a new and direct biological connection between light and PDEs. Light-regulated PDEs are under development, and of demand to complete the toolkit for cAMP/cGMP manipulation. In this review, we summarize the state of the art, pros and cons of artificial and natural light-regulated PDEs, and discuss potential new strategies of developing light-gated PDEs for optogenetic manipulation.

(1) Background: After the discovery and application of Chlamydomonas reinhardtii channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca\(^{2+}\) conductance or more specific ion permeability are in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos and PsChR and compared them with published ChR2 aspartate mutants. (3) Results: We found that the ChR2 D156H mutant (XXM) showed enhanced Na\(^+\) and Ca\(^{2+}\) conductance, which was not noticed before, while the D156C mutation (XXL) influenced the Na\(^+\) and Ca\(^{2+}\) conductance only slightly. The aspartate to histidine and cysteine mutations of Chronos and PsChR also influenced their photocurrent, ion permeability, kinetics, and light sensitivity. Most interestingly, PsChR D139H showed a much-improved photocurrent, compared to wild type, and even higher Na+ selectivity to H\(^+\) than XXM. PsChR D139H also showed a strongly enhanced Ca\(^{2+}\) conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the TM4 influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na\(^+\) selectivity and Ca\(^{2+}\) conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for Ca\(^{2+}\) manipulation.

Optogenetics is a method to control the cell activity with light by expression of a natural or engineered photoreceptor via genetic modification technology. Optogenetics early success came with the light-gated cation channel "Channelrhodopsin-2" in neurons and expanded from neuroscience to other research fields such as cardiac research and cell signaling, also due to the enrichment by new photoreceptors. In this study, I focus on searching and characterizing new photoreceptors to expand the optogenetic tool box. In this work I characterize three newly discovered microbial rhodopsins and some engineered mutants of them. The first rhodopsin is a proton pump from the diatom Fragilariopsis cylindrus, Fragilariopsis Rhodopsin or abbreviated: FR. I cloned the full-length FR and proved it to be a light-activated proton pump with high efficacy in comparison to Bacteriorhodopsin (BR). During this study, I also developed a new method to improve the plasma membrane targeting of several microbial rhodopsins. I also obtained a FR mutant (channel-like FR or chFR) which behaves like a light-gated proton channel. FR can be used for optogenetic hyperpolarization or alkalization of a cell while the chFR could be used for depolarization or lowering of the cellular pH. The induction of FR expression under iron-limited conditions in the diatom indicated an alternative energy generation mechanism of F. cylindrus when iron-containing enzymes are scarce. I then characterized a new microbial rhodopsin with novel light-regulated Guanylyl Cyclase (GC) activity. This rhodopsin guanylyl cyclase from the fungus Blastocladiella emersonii (B.e. CyclaseOpsin or BeCyclOp) has been proven by me to be an efficient light-gated GC with high specificity and fast kinetics. BeCyclOp also has a novel structure with eight transmembrane helices, containing a long cytosolic N-terminus which participates in the tight regulation of the GC activity. In collaboration with Prof. Alexander Gottschalk (Univ. Frankfurt/M.), BeCyclOp has been tested in muscle cells and sensory neurons of Caenorhabditis elegans and proven to be a powerful optogenetic tool in a living animal. I also generated a BeCyclOp mutant with enhanced light sensitivity. Already more than ten years ago, guanylyl cyclase rhodopsins were suggested to exist in Chlamydomonas reinhardtii by analyzing genomic sequence data. But until now no functional proof existed. By further cloning and sequencing I discovered such a rhodopsin with light-regulated guanylyl cyclase activity. This functional Cyclaseopsin (COP6c) is quite different to BeCyclOp, as it was proven to be a light-inhibited GC. Cop6c is much larger than BeCyclOp with a His-Kinase and a response regulator domain between the rhodopsin and the cyclase domain. I also introduced a new strategy for generating optogenetic tools by fusing the photoactivated adenylyl cyclase bPAC to two different CNG channels. These new tools function via light-gated cAMP production and subsequent CNG channel activation. These tools combined the properties of bPAC (highly sensitive to blue light) and CNG channels (high single-channel conductance and high Ca2+ permeability), as demonstrated by expression in Xenopus oocytes. As a further benefit the fusing of bPAC to CNG channels leads to a bPAC with a more than tenfold reduced dark activity which is a valuable improvement for bPAC itself as an optogenetic tool.

Background Microbial rhodopsins vary in their chemical properties, from light sensitive ion transport to different enzymatic activities. Recently, a novel family of two-component Cyclase (rhod)opsins (2c-Cyclop) from the green algae Chlamydomonas reinhardtii and Volvox carteri was characterized, revealing a light-inhibited guanylyl cyclase (GC) activity. More genes similar to 2c-Cyclop exist in algal genomes, but their molecular and physiological functions remained uncharacterized. Results Chlamyopsin-5 (Cop5) from C. reinhardtii is related to Cr2c-Cyclop1 (Cop6) and can be expressed in Xenopus laevis oocytes, but shows no GC activity. Here, we exchanged parts of Cop5 with the corresponding ones of Cr2c-Cyclop1. When exchanging the opsin part of Cr2c-Cyclop1 with that of Cop5, we obtained a bi-stable guanylyl cyclase (switch-Cyclop1) whose activity can be switched by short light flashes. The GC activity of switch-Cyclop1 is increased for hours by a short 380 nm illumination and switched off (20-fold decreased) by blue or green light. switch-Cyclop1 is very light-sensitive and can half-maximally be activated by ~ 150 photons/nm2 of 380 nm (~ 73 J/m2) or inhibited by ~ 40 photons/nm\(^2\) of 473 nm (~ 18 J/m\(^2\)). Conclusions This engineered guanylyl cyclase is the first light-switchable enzyme for cGMP level regulation. Light-regulated cGMP production with high light-sensitivity is a promising technique for the non-invasive investigation of the effects of cGMP signaling in many different tissues.