Channelrhodopsin-1 Phosphorylation Changes with Phototactic Behavior and Responds to Physiological Stimuli in Chlamydomonas.

The unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) exhibits oriented movement responses (phototaxis) to light over more than three log units of intensity. Phototaxis thus depends on the cell's ability to adjust the sensitivity of its photoreceptors to ambient light conditions. In Chlamydomonas, the photoreceptors for phototaxis are the channelrhodopsins (ChR)1 and ChR2; these light-gated cation channels are located in the plasma membrane. Although ChRs are widely used in optogenetic studies, little is known about ChR signaling in algae. We characterized the in vivo phosphorylation of ChR1. Its reversible phosphorylation occurred within seconds as a graded response to changes in the light intensity and ionic composition of the medium and depended on an elevated cytosolic Ca2+ concentration. Changes in the phototactic sign were accompanied by alterations in the phosphorylation status of ChR1. Furthermore, compared with the wild type, a permanently negative phototactic mutant required higher light intensities to evoke ChR1 phosphorylation. C-terminal truncation of ChR1 disturbed its reversible phosphorylation, whereas it was normal in ChR2-knockout and eyespot-assembly mutants. The identification of phosphosites in regions important for ChR1 function points to their potential regulatory role(s). We propose that multiple ChR1 phosphorylation, regulated via a Ca2+-based feedback loop, is an important component in the adaptation of phototactic sensitivity in Chlamydomonas.

The intrinsic amyloidogenic propensity of cofilin-1 is aggravated by Cys-80 oxidation: A possible link with neurodegenerative diseases.

Cofilin-1, an actin dynamizing protein, forms actin-cofilin rods, which is one of the major events that exacerbates the pathophysiology of amyloidogenic diseases. Cysteine oxidation in cofilin-1 under oxidative stress plays a crucial role in the formation of these rods. Others and we have reported that cofilin-1 possesses a self-oligomerization property in vitro and in vivo under physiological conditions. However, it remains elusive if cofilin-1 itself forms amyloid-like structures. We, therefore, hypothesized that cofilin-1 might form amyloid-like assemblies, with a potential to intensify the pathophysiology of amyloid-linked diseases. We used various in silico and in vitro techniques and examined the amyloid-forming propensity of cofilin-1. The study confirms that cofilin-1 possesses an intrinsic tendency of aggregation and forms amyloid-like structures in vitro. Further, we studied the effect of cysteine oxidation on the stability and structural features of cofilin-1. Our data show that oxidation at Cys-80 renders cofilin-1 unstable, leading to a partial loss of protein structure. The results substantiate our hypothesis and establish a strong possibility that cofilin-1 aggregation might play a role in cofilin-mediated pathology and the progression of several amyloid-linked diseases.

Centriole is the pivot coordinating dynamic signaling for cell proliferation and organization during early development in the vertebrates.

Vertebrates have an elaborate and functionally segmented body. It evolves from a single cell by systematic cell proliferation but attains a complex body structure with exquisite precision. This development requires two cellular events: cell cycle and ciliogenesis. For these events, the dynamic molecular signaling is converged at the centriole. The cell cycle helps in cell proliferation and growth of the body and is a highly regulated and integrated process. Its errors cause malignancies and developmental disorders. The cells newly proliferated are organized during organogenesis. For a cellular organization, dedicated signaling hubs are developed in the cells, and most often cilia are utilized. The cilium is generated from one of the centrioles involved in cell proliferation. The developmental signaling pathways hosted in cilia are essential for the elaboration of the body plan. The cilium's compartmental seclusion is ideal for noise-free molecular signaling and is essential for the precision of the body layout. The dysfunctional centrioles and primary cilia distort the development of body layout that manifest as serious developmental disorders. Thus, centriole has a dual role in the growth and cellular organization. It organizes dynamically expressed molecules of cell cycle and ciliogenesis and plays a balancing act to generate new cells and organize them during development. A putative master molecule may regulate and coordinate the dynamic gene expression at the centrioles. The convergence of many critical signaling components at the centriole reiterates the idea that centriole is a major molecular workstation involved in elaborating the structural design and complexity in vertebrates. This article is protected by copyright. All rights reserved.

Modulation of Phototropin Signalosome with Artificial Illumination Holds Great Potential in the Development of Climate-Smart Crops.

Changes in environmental conditions like temperature and light critically influence crop production. To deal with these changes, plants possess various photoreceptors such as Phototropin (PHOT), Phytochrome (PHY), Cryptochrome (CRY), and UVR8 that work synergistically as sensor and stress sensing receptors to different external cues. PHOTs are capable of regulating several functions like growth and development, chloroplast relocation, thermomorphogenesis, metabolite accumulation, stomatal opening, and phototropism in plants. PHOT plays a pivotal role in overcoming the damage caused by excess light and other environmental stresses (heat, cold, and salinity) and biotic stress. The crosstalk between photoreceptors and phytohormones contributes to plant growth, seed germination, photo-protection, flowering, phototropism, and stomatal opening. Molecular genetic studies using gene targeting and synthetic biology approaches have revealed the potential role of different photoreceptor genes in the manipulation of various beneficial agronomic traits. Overexpression of PHOT2 in Fragaria ananassa leads to the increase in anthocyanin content in its leaves and fruits. Artificial illumination with blue light alone and in combination with red light influence the growth, yield, and secondary metabolite production in many plants, while in algal species, it affects growth, chlorophyll content, lipid production and also increases its bioremediation efficiency. Artificial illumination alters the morphological, developmental, and physiological characteristics of agronomic crops and algal species. This review focuses on PHOT modulated signalosome and artificial illumination-based photo-biotechnological approaches for the development of climate-smart crops.

Genome-wide identification and biochemical characterization of calcineurin B-like calcium sensor proteins in Chlamydomonas reinhardtii.

Calcium (Ca2+) signaling is involved in the regulation of diverse biological functions through association with several proteins that enable them to respond to abiotic and biotic stresses. Though Ca2+-dependent signaling has been implicated in the regulation of several physiological processes in Chlamydomonas reinhardtii, Ca2+ sensor proteins are not characterized completely. C. reinhardtii has diverged from land plants lineage, but shares many common genes with animals, particularly those encoding proteins of the eukaryotic flagellum (or cilium) along with the basal body. Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is an important effector of Ca2+ signaling in animals, while calcineurin B-like proteins (CBLs) play an important role in Ca2+ sensing and signaling in plants. The present study led to the identification of 13 novel CBL-like Ca2+ sensors in C. reinhardtii genome. One of the archetypical genes of the newly identified candidate, CrCBL-like1 was characterized. The ability of CrCBL-like1 protein to sense as well as bind Ca2+ were validated using two-step Ca2+-binding kinetics. The CrCBL-like1 protein localized around the plasma membrane, basal bodies and in flagella, and interacted with voltage-gated Ca2+ channel protein present abundantly in the flagella, indicating its involvement in the regulation of the Ca2+ concentration for flagellar movement. The CrCBL-like1 transcript and protein expression were also found to respond to abiotic stresses, suggesting its involvement in diverse physiological processes. Thus, the present study identifies novel Ca2+ sensors and sheds light on key players involved in Ca2+signaling in C. reinhardtii, which could further be extrapolated to understand the evolution of Ca2+ mediated signaling in other eukaryotes.

Penta- and hexa-coordinate ferric hemoglobins display distinct pH titration profiles measured by Soret peak shifts.

Hemoglobins with diverse characteristics have been identified in all kingdoms of life. Their ubiquitous presence indicates that these proteins play important roles in physiology, though function for all hemoglobins are not yet established with certainty. Their physiological role may depend on their ability to bind ligands, which in turn is dictated by their heme chemistry. However, we have an incomplete understanding of the mechanism of ligand binding for these newly discovered hemoglobins and the measurement of their kinetic parameters depend on their coordination at the heme iron. To gain insights into their functional role, it is important to categorize the new hemoglobins into either penta- or hexa-coordinated varieties. We demonstrate that simple pH titration and absorbance measurements can determine the coordination state of heme iron atom in ferric hemoglobins, thus providing unambiguous information about the classification of new globins. This method is rapid, sensitive and requires low concentration of protein. Penta- and hexa-coordinate hemoglobins displayed distinct pH titration profiles as observed in a variety of hemoglobins. The pentacoordinate distal histidine mutant proteins of hexacoordinate hemoglobins and ligand-bound hexacoordinate forms of pentacoordinate hemoglobins reverse the pH titration profiles, thus validating the sensitivity of this spectroscopic technique.

Phototropin influence on eyespot development and regulation of phototactic behavior in Chlamydomonas reinhardtii.

The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic orientation of the alga. Here, we found that eyespot size is strain specific and downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted by homologous recombination, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal light, oxygen or voltage sensing domains (LOV1+LOV2) alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. Moreover, phototropin is involved in adjusting the level of channelrhodopsin-1, the dominant primary receptor for phototaxis within the eyespot. Both the level of channelrhodopsin-1 at the onset of illumination and its steady state level during the light period are downregulated by phototropin, whereas the level of channelrhodopsin-2 is not significantly altered. Furthermore, a light intensity-dependent formation of a C-terminal truncated phototropin form was observed. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase.

Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases.

The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Our approach includes (i) design of gene-specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3'-phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin-resistant (Pm-R) clones with expressing nucleases. Of these Pm-R clones, 1% also contained a modified COP3 locus. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.

Cellular organelles facilitate dimerization of a newly identified Arf from Chlamydomonas reinhardtii.

GTPases of the Ras superfamily regulate a wide variety of cellular processes including vesicular transport and various secretory pathways of the cell. ADP - ribosylation factor (ARF) belongs to one of the five major families of the Ras superfamily and serves as an important component of vesicle formation and transport machinery of the cells. The binding of GTP to these Arfs and its subsequent hydrolysis, induces conformational changes in these proteins leading to their enzymatic activities. The dimeric form of Arf is associated with membrane pinch-off during vesicle formation. In this report, we have identified an arf gene from the unicellular green alga Chlamydomonas reinhardtii, CrArf, and showed that the oligomeric state of the protein in C. renhardtii is modulated by the cellular membrane environment of the organism. Protein cross-linking experiments showed that the purified recombinant CrArf has the ability to form a dimer. Both the 20-kDa monomeric and 40-kDa dimeric forms of CrArf were recognized from Chlamydomonas total cell lysate (CrTLC) and purified recombinant CrArf by the CrArf specific antibody. The membranous environment of the cell appeared to facilitate dimerization of the CrArf, as dimeric form was found exclusively associated with the membrane bound organelles. The subcellular localization studies in Chlamydomonas suggested that CrArf mainly localized in the cytosol and was mislocalized in vesicle transport machinery inhibitor treated cells. This research sheds light on the importance of the cellular membrane environment for regulating the oligomeric state of CrArf protein in this organism and associated functional role.

A photochromic histidine kinase rhodopsin (HKR1) that is bimodally switched by ultraviolet and blue light.

Rhodopsins are light-activated chromoproteins that mediate signaling processes via transducer proteins or promote active or passive ion transport as ion pumps or directly light-activated channels. Here, we provide spectroscopic characterization of a rhodopsin from the Chlamydomonas eyespot. It belongs to a recently discovered but so far uncharacterized family of histidine kinase rhodopsins (HKRs). These are modular proteins consisting of rhodopsin, a histidine kinase, a response regulator, and in some cases an effector domain such as an adenylyl or guanylyl cyclase, all encoded in a single protein as a two-component system. The recombinant rhodopsin fragment, Rh, of HKR1 is a UVA receptor (λ(max) = 380 nm) that is photoconverted by UV light into a stable blue light-absorbing meta state Rh-Bl (λ(max) = 490 nm). Rh-Bl is converted back to Rh-UV by blue light. Raman spectroscopy revealed that the Rh-UV chromophore is in an unusual 13-cis,15-anti configuration, which explains why the chromophore is deprotonated. The excited state lifetime of Rh-UV is exceptionally stable, probably caused by a relatively unpolar retinal binding pocket, converting into the photoproduct within about 100 ps, whereas the blue form reacts 100 times faster. We propose that the photochromic HKR1 plays a role in the adaptation of behavioral responses in the presence of UVA light.

Effect of tryptophan mutation on the structure of LOV1 domain of phototropin1 protein of Ostreococcus tauri: A combined molecular dynamics simulation and biophysical approach.

BACKGROUND: Light, oxygen and voltage (LOV) proteins detect blue light by formation of a covalent 'photoadduct' between the flavin chromophore and the neighboring conserved cysteine residue. LOV proteins devoid of this conserved photoactive cysteine are unable to form this 'photoadduct' upon light illumination, but they can still elicit functional response via the formation of neutral flavin radical. Recently, tryptophan residue has been shown to be the primary electron donors to the flavin excited state. METHODS: Photoactive cysteine (Cys42) and tryptophan (Trp68) residues in the LOV1 domain of phototropin1 of Ostreococcus tauri (OtLOV1) was mutated to alanine and threonine respectively. Effect of these mutations have been studied using molecular dynamics simulation and spectroscopic techniques. RESULTS: Molecular dynamics simulation indicated that W68T did not affect the structure of OtLOV1 protein, but C42A leads to some structural changes. An increase in the fluorescence lifetime and quantum yield values was observed for the Trp68 mutant. CONCLUSIONS: An increase in the fluorescence lifetime and quantum yield of Trp68 mutant compared to the wild type protein suggests that Trp68 residue participates in quenching of the flavin excited state followed by photoexcitation. GENERAL SIGNIFICANCE: Enhanced photo-physical properties of Trp68 OtLOV1 mutant might enable its use for the optogenetic and microscopic applications.

Algal rhodopsins encoding diverse signal sequence holds potential for expansion of organelle optogenetics.

Rhodopsins have been extensively employed for optogenetic regulation of bioelectrical activity of excitable cells and other cellular processes across biological systems. Various strategies have been adopted to attune the cellular processes at the desired subcellular compartment (plasma membrane, endoplasmic reticulum, Golgi, mitochondria, lysosome) within the cell. These strategies include-adding signal sequences, tethering peptides, specific interaction sites, or mRNA elements at different sites in the optogenetic proteins for plasma membrane integration and subcellular targeting. However, a single approach for organelle optogenetics was not suitable for the relevant optogenetic proteins and often led to the poor expression, mislocalization, or altered physical and functional properties. Therefore, the current study is focused on the native subcellular targeting machinery of algal rhodopsins. The N- and C-terminus signal prediction led to the identification of rhodopsins with diverse organelle targeting signal sequences for the nucleus, mitochondria, lysosome, endosome, vacuole, and cilia. Several identified channelrhodopsins and ion-pumping rhodopsins possess effector domains associated with DNA metabolism (repair, replication, and recombination) and gene regulation. The identified algal rhodopsins with diverse effector domains and encoded native subcellular targeting sequences hold immense potential to establish expanded organelle optogenetic regulation and associated cellular signaling.

Multiomics and optobiotechnological approaches for the development of microalgal strain for production of aviation biofuel and biorefinery.

Demand and consumption of fossil fuels is increasing daily, and oil reserves are depleting. Technological developments are required towards developing sustainable renewable energy sources and microalgae are emerging as a potential candidate for various application-driven research. Molecular understanding attained through omics and system biology approach empowering researchers to modify various metabolic pathways of microalgal system for efficient extraction of biofuel and important biomolecules. This review furnish insight into different "advanced approaches" like optogenetics, systems biology and multi-omics for enhanced production of FAS (Fatty Acid Synthesis) and lipids in microalgae and their associated challenges. These new approaches would be helpful in the path of developing microalgae inspired technological platforms for optobiorefinery, which could be explored as source material to produce biofuels and other valuable bio-compounds on a large scale.

Characterization of Chlamydomonas voltage-gated calcium channel and its interaction with photoreceptor support VGCC modulated photobehavioral response in the green alga.

Calcium (Ca2+) signaling plays a major role in regulating multiple processes in living cells. The photoreceptor potential in Chlamydomonas triggers the generation of all or no flagellar Ca2+ currents that cause membrane depolarization across the eyespot and flagella. Modulation in membrane potential causes changes in the flagellar waveform, and hence, alters the beating patterns of Chlamydomonas flagella. The rhodopsin-mediated eyespot membrane potential is generated by the photoreceptor Ca2+ current or P-current however, the flagellar Ca2+ currents are mediated by unidentified voltage-gated calcium (VGCC or CaV) and potassium channels (VGKC). The voltage-gated ion channel that associates with ChRs to generate Ca2+ influx across the flagella and its cellular distribution has not yet been identified. Here, we identified putative VGCCs from algae and predicted their novel properties through insilico analysis. We further present experimental evidence on Chlamydomonas reinhardtii VGCCs to predict their novel physiological roles. Our experimental evidences showed that CrVGCC4 localizes to the eyespot and flagella of Chlamydomonas and associates with channelrhodopsins (ChRs). Further in silico interactome analysis of CrVGCCs suggested that they putatively interact with photoreceptor proteins, calcium signaling, and intraflagellar transport components. Expression analysis indicated that these VGCCs and their putative interactors can be perturbed by light stimuli. Collectively, our data suggest that VGCCs in general, and VGCC4 in particular, might be involved in the regulation of the photobehavioral response of Chlamydomonas.

Deciphering the role of cytoplasmic domain of Channelrhodopsin in modulation of the interactome and SUMOylome of Chlamydomonas reinhardtii.

Translocation of channelrhodopsins (ChRs) is mediated by the intraflagellar transport (IFT) machinery. However, the functional role of the network involving photoreceptors, IFT and other proteins in controlling algal ciliary motility is still not fully delineated. In the current study, we have identified two important motifs at the C-terminus of ChR1, VXPX and LKNE. VXPX is a known ciliary targeting sequence in animals, and LKNE is a well-known SUMOylation motif. To the best of our knowledge, this study gives prima facie insight into the role of SUMOylation in Chlamydomonas. We prove that VMPS of ChR1 is important for interaction with GTPase CrARL11. We show that SUMO motifs are present in the C-terminus of putative ChR1s from green algae. Performing experiments with n-Ethylmaleimide (NEM) and Ubiquitin-like protease 1 (ULP-1), we show that SUMOylation may modulate ChR1 protein in Chlamydomonas. Experiments with 2D08, a known sumoylation blocker, increased the concentration of ChR1 protein. Finally, we show the endogenous SUMOylated proteins (SUMOylome) of C. reinhardtii, identified by using immunoprecipitation followed by nano-LC-MS/MS detection. This report establishes a link between evolutionarily conserved SUMOylation and ciliary machinery for the maintenance and functioning of cilia across the eukaryotes. Our enriched SUMOylome of C. reinhardtii comprehends the proteins related to ciliary development and photo-signaling, along with the orthologue(s) associated to human ciliopathies as SUMO targets.

Role of calcium sensor protein module CBL-CIPK in abiotic stress and light signaling responses in green algae.

Ca2+ signaling is an important biological process that enable to perceive and communicate information in the cell. Our current understanding of the signaling system suggests that plants and animals have certain differences in signal-sensing mechanisms. The Ca2+-mediated CBL-CIPK module has emerged as a major sensor responder network for Ca2+ signaling and has been speculated to be involved in plant terrestrial life adaptation. This module has previously been reported in Archaeplastids, Chromalveolates, and Excavates. In our experimental analysis of Chlamydomonas reinhardtii CBLs, we proved that the CrCBL1 protein interacts with Phototropin and Channelrhodopsin, and the expression of CrCBLs is modulated by light. Further analysis using chlorophyte and streptophyte algal sequences allowed us to identify the differences that have evolved in CBL and CIPK proteins since plants have progressed from aquatic to terrestrial habitats. Moreover, an investigation of Klebsormidium CBL and CIPK genes led us to know that they are abiotic stress stimuli-responsive, indicating that their role was defined very early during terrestrial adaptations. Structure-based prediction and Ca2+-binding assays indicated that the KnCBL1 protein in Klebsormidium showed a typical Ca2+-binding pocket. In summary, the results of this study suggest that these stress-responsive proteins enable crosstalk between Ca2+ and light signaling pathways very early during plant adaptation from aquatic to terrestrial habitats.

COVID-19 ORF3a Viroporin-Influenced Common and Unique Cellular Signaling Cascades in Lung, Heart, and the Brain Choroid Plexus Organoids with Additional Enriched MicroRNA Network Analyses for Lung and the Brain Tissues.

Tissue-specific implications of SARS-CoV-2-encoded accessory proteins are not fully understood. SARS-CoV-2 infection can severely affect three major organs-the heart, lungs, and brain. We analyzed SARS-CoV-2 ORF3a interacting host proteins in these three major organs. Furthermore, we identified common and unique interacting host proteins and their targeting miRNAs (lung and brain) and delineated associated biological processes by reanalyzing RNA-seq data from the brain (COVID-19-infected/uninfected choroid plexus organoid study), lung tissue from COVID-19 patients/healthy subjects, and cardiomyocyte cells-based transcriptomics analyses. Our in silico studies showed ORF3a interacting proteins could vary depending upon tissues. The number of unique ORF3a interacting proteins in the brain, lungs, and heart were 10, 7, and 1, respectively. Though common pathways influenced by SARS-CoV-2 infection were more, unique 21 brain and 7 heart pathways were found. One unique pathway for the heart was negative regulation of calcium ion transport. Reported observations of COVID-19 patients with a history of hypertension taking calcium channel blockers (CCBs) or dihydropyridine CCBs had an elevated rate of intubation or increased rate of intubation/death, respectively. Also, the likelihood of hospitalization of chronic CCB users with COVID-19 was greater in comparison to long-term angiotensin-converting enzyme inhibitors/angiotensin receptor blockers users. Further studies are necessary to confirm this. miRNA analysis of ORF3a interacting proteins in the brain and lungs revealed 3 of 37 brain miRNAs and 1 of 25 lung miRNAs with high degree and betweenness indicating their significance as hubs in the interaction network. Our study could help in identifying potential tissue-specific COVID-19 drug/drug repurposing targets.

Biomanufacturing of glycosylated antibodies: Challenges, solutions, and future prospects.

Traditionally, recombinant protein production has been done in several expression hosts of bacteria, fungi, and majorly CHO (Chinese Hamster Ovary) cells; few have high production costs and are susceptible to harmful toxin contamination. Green algae have the potential to produce recombinant proteins in a more sustainable manner. Microalgal diversity leads to offer excellent opportunities to produce glycosylated antibodies. An antibody with humanized glycans plays a crucial role in cellular communication that works to regulate cells and molecules, to control disease, and to stimulate immunity. Therefore, it becomes necessary to understand the role of abiotic factors (light, temperature, pH, etc.) in the production of bioactive molecules and molecular mechanisms of product synthesis from microalgae which would lead to harnessing the potential of algal bio-refinery. However, the potential of microalgae as the source of bio-refinery has been less explored. In the present review, omics approaches for microalgal engineering, methods of humanized glycoproteins production focusing majorly on N-glycosylation pathways, light-based regulation of glycosylation machinery, and production of antibodies with humanized glycans in microalgae with a major emphasis on modulation of post-translation machinery of microalgae which might play a role in better understanding of microalgal potential as a source for antibody production along with future perspectives.

A conserved isoleucine in the LOV1 domain of a novel phototropin from the marine alga Ostreococcus tauri modulates the dark state recovery of the domain.

BACKGROUND: Phototropins are UV-A/blue light receptor proteins with two LOV (Light-Oxygen-Voltage) sensor domains at their N terminus and a kinase domain at the C-terminus in photoautotrophic organisms. This is the first research report of a canonical phototropin from marine algae Ostreococcus tauri. METHODS: We synthesized core LOV1 (OtLOV1) domain-encoding portion of the phototropin gene of O. tauri, the domain was heterologously expressed, purified and assessed for its spectral properties and dark recovery kinetics by UV-Visible, fluorescence spectroscopy and mutational studies. Quaternary structure characteristics were studied by SEC and glutaraldehyde crosslinking. RESULTS: The absorption spectrum of OtLOV1 lacks the characteristic 361nm peak shown by other LOV1 domains. It undergoes a photocycle with a dark state recovery time of approximately 30min (τ=300.35s). Native OtLOV1 stayed as dimer in aqueous solution and the dimer formation was light and concentration independent. Mutating isoleucine at 43rd position to valine accelerated the dark recovery time by more than 10-fold. Mutating it to serine reduced sensitivity to blue light, but the dark recovery time remained unaltered. I43S mutation also destabilized the FMN binding to a great extent. CONCLUSION: The OtLOV1 domain of the newly identified OtPhot is functional and the isoleucine at position 43 of OtLOV1 is the key residue responsible for fine-tuning the domain properties. GENERAL SIGNIFICANCE: This is the first characterized LOV1 domain of a canonical phototropin from a marine alga and spectral properties of the domain are similar to that of the LOV1 domain of higher plants.

Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles.

BACKGROUND: Elucidation of molecular mechanism of silver nanoparticles (SNPs) biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution) of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. RESULTS: The C. reinhardtii cell free extract (in vitro) and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro) SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP⁺ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. CONCLUSION: Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver nanoparticles using C. reinhardtii as a model system.