Anabaena Sensory Rhodopsin Transducer Research Articles

Kinetics of DNA looping by Anabaena sensory rhodopsin transducer (ASRT) by using DNA cyclization assay

DNA cyclization assay together with single-molecule FRET was employed to monitor protein-mediated bending of a short dsDNA (~ 100 bp). This method provides a simple and easy way to monitor the structural change of DNA in real-time without necessitating prior knowledge of the molecular structures for the optimal dye-labeling. This assay was applied to study how Anabaena sensory rhodopsin transducer (ASRT) facilitates loop formation of DNA as a possible mechanism for gene regulation. The ASRT-induced DNA looping was maximized at 50 mM of Na+, while Mg2+ also played an essential role in the loop formation.

Receptor Binding and Phosphorylation of Anabaena Sensory Rhodopsin Transducer towards Signaling via Protein‐Protein Cross Talk

Anabaena sensory rhodopsin transducer, ASRT [125 amino acid sequence] is present under the same promoter of its sensory rhodopsin, ASR in Anabaena PCC 7120 a freshwater, filamentous, photosynthetic cynobacteria. The ASRT exists as tetrameric soluble protein indicated to function as downstream signaling partner to sensory rhodopsin. Both solution and crystal structures has reveled that this primarily beta stranded protein exhibit a helical face at carboxyl terminus. Besides receptor binding, ASRT is been shown to serve as a novel eukaryotic-like interaction with DNA. We have demonstrated that carboxyl helical face of tetramer is involved in unusual tetrameric stability. A blast search matches the ASRT to others of unknown function, with 84% similarity and 74% identity to a protein in the myxobacterium Polyangium cellulosum and with 72% similarity and 58% identity to a protein in the legume symbiont Sinorhizobium meliloti. Often the homologues of this family are termed as “DUF”, Domain of Unknown Function member. This DUF1362 family is wide spread over microbial population including pathogens as well. The gene neighbor hood, along with structural fold analysis termed this family as novel small carbohydrate binding protein. We have observed unique xylan binding via electrostatic interaction. Additional bioinformatics analysis has revealed a phosphor transfer along with unique structural fold that may transform it as a unique carbohydrate binding module. However, the signaling state/mechanism of ASRT is obscure. Our initial data with efficient donor, acetyl phosphate, acP using fluorescence quenching of lone Trp-9 suggests that ASRT is involved in phosphorylation. We noticed two notable consensus phosphor transfer residue motif in ASRT, SDKE [53–56] and TRLD [105–108]. Acetyl phosphate has been used as phosphoryl donor in vitro to numerous response regulators [CheY/PhoB/OmpR]. The extent of quenching in presence of Mg2+by increasing concentration of acP yielded lower KM [~21mM] compared to PhoB [~8mM]. It is likely that proximity of phosphor accepting residue (s) to the lone Trp is not comparable to others. Interestingly the ASRT mutant E56Q and D108N indicate the loss of phosphor transfer. We hypothesize that putative position shown as red in figure is involved in ASRT phosphorylation. This motif is in close proximity to receptor binding deduced by CABS docking and GST-assay using various length of ASR [full length, 1–261, and truncated (1–229, 1–239, 1–249)]. Phosphor transfer data along with plausible impact on tetrameric stability will be discussed in this study. We have identified a Serine Threonine Kinase, STK 3169 gene as a likely candidate in phosphor relay. Recent results of STK 3169 cloning, optimized expression, purification and subsequent characterization in phosphorylation assay will be presented. Support or Funding Information Supported by NIH-NIGMS Support for Competitive Research, SCORE SC3 award GM113803 to VT This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

“Ligand Binding and Phosphorylation of Anabaena Sensory Rhodopsin Transducer towards Putative Signaling State in Sensory Rhodopsin Mediated Protein‐Protein Cross Talk”

Anabaena sensory rhodopsin transducer, ASRT [125 amino acid sequence] is present under the same promoter of its sensory rhodopsin, ASR in Anabaena PCC 7120 a freshwater, filamentous, photosynthetic cynobacteria. The ASRT exists as tetrameric soluble protein indicated to function as downstream signaling partner to sensory rhodopsin. Both solution and crystal structures has reveled that this primarily beta stranded protein exhibit a helical face at carboxyl terminus. Besides receptor binding, ASRT is been shown to serve as a novel eukaryotic-like interaction with DNA. We have demonstrated that carboxyl helical face of tetramer is involved in unusual tetrameric stability. A blast search matches the ASRT to others of unknown function, with 84% similarity and 74% identity to a protein in the myxobacterium Polyangium cellulosum and with 72% similarity and 58% identity to a protein in the legume symbiont Sinorhizobium meliloti. Often the homologues of this family are termed as “DUF”, Domain of Unknown Function member. This DUF1362 family is wide spread over microbial population including pathogens as well. The gene neighbor hood, along with structural fold analysis termed this family as novel small carbohydrate binding protein. We have observed unique xylan binding via electrostatic interaction. Additional bioinformatics analysis has revealed a phosphor transfer along with unique structural fold that may transform it as a unique carbohydrate binding module. However, the signaling state/mechanism of ASRT is obscure. Our initial data with efficient donor, acetyl phosphate, acP using fluorescence quenching of lone Trp-9 suggests that ASRT is involved in phosphorylation. We noticed two notable consensus phosphor transfer residue motif in ASRT, SDKE [53–56] and TRLD [105–108]. Acetyl phosphate has been used as phosphoryl donor in vitro to numerous response regulators [CheY/PhoB/OmpR]. The extent of quenching in presence of Mg2+ by increasing concentration of acP yielded lower KM [~21 mM] compared to PhoB [~8 mM]. It is likely that proximity of phosphor accepting residue (s) to the lone Trp is not comparable to others. Interestingly the ASRT mutant E56Q and D108N indicate the loss of phosphor transfer. We hypothesize that putative position shown as red in figure is involved in ASRT phosphorylation. This motif is in close proximity to receptor binding deduced by CABS docking. Phosphor transfer data along with plausible impact on tetrameric stability will be discussed in this study. We have identified a Serine Threonine Kinase, STK 3169 gene as a likely candidate in phosphor relay. We have cloned and optimizing the functional expression, purification for subsequent characterization in phosphorylation assay. Support or Funding Information Supported by NIH-NIGMS SCORE SC3GM113803 & BCU CURE 2017 award to VT Helical Face of ASRT with Phosphor Transfer Site This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bending of Short dsDNA Upon Binding of Anabaena Sensory Rhodopsin Transducer

Single-molecule fluorescence resonance energy transfer (smFRET) has been used to monitor the intra-molecular changes of DNA or proteins. Practically, the structure information of the macromolecule is required to precisely label the molecules with two fluorescence dyes. The structure information is, however, often not available, which limits the FRET technique from application. In this study, we applied a recently developed single-molecule cyclization assay, to monitor the structural change of double stranded DNA upon binding of Anabaena sensory rhodopsin transducer (ASRT), a novel signal transducer protein involved in photo-sensory and light harvesting system in bacteria whose DNA-bound structure is unknown. We first labeled the two ends of a linear double stranded DNA with a FRET pair, from which the distance between the two ends can be estimated after binding of ASRT. However, FRET would occur only if the two ends are in the FRET range (typically a few nanometers), which cannot be guaranteed without given the structure. To overcome this problem, we placed complementary single stranded tails at the two ends of the DNA, so that the tails may interact and bind to each other and bring the two ends in close proximity. We observed the enhanced loop formation of DNA (∼100 bp) upon binding of ASRT, which suggests that ASRT regulates gene expression by facilitating loop formation of the dsDNA around itself.

Backbone assignments of Anabaena Sensory Rhodopsin Transducer with DNA

Backbone assignments of Anabaena Sensory Rhodopsin Transducer with DNA

A Eukaryotic-Like Interaction of Soluble Cyanobacterial Sensory Rhodopsin Transducer with DNA

Anabaena sensory rhodopsin is a recently discovered membrane photosensor with a unique signal transduction cascade. It interacts with a soluble tetrameric transducer [ Anabaena sensory rhodopsin transducer (ASRT)] that can bind to promoter regions of several genes related to the utilization of light energy. Even though the X-ray crystal structure of ASRT is available, the mechanism of its interaction with DNA is still unknown. We used solution NMR to understand the mechanism of the DNA binding. Both X-ray crystal structures and solution NMR data reveal seven β-strands forming a rigid scaffold (β-face) and a flexible, partially disordered α-face, comprised by the C-termini and loops. We found that the conformation of the α-face in solution is very different from that in the crystals. While the C-termini of crystalline ASRT are solvent exposed and either α-helical or disordered, about half of ASRT monomers in solution feature buried C-terminal β-strand, with another half of C-tails being random coils. Titration of ASRT with a 20-bp fragment of the pec operon promoter showed that only monomers with β-structured C-tails bind the DNA. NMR signals suggest that specific Arg and Asn/Gln residues are involved in the interaction with DNA. The DNA binding occurs with micromolar affinity and a 1:1 stoichiometry (DNA:ASRT tetramer) and results in a significant ordering of the α-face involving the extension of the C-terminal β-strand and reorganization of the first loop. Such induced-fit type of interaction, which mainly utilizes loops between β-strands and results in the increase in their order, is typical for eukaryotic transcription factors of the immunoglobulin-like fold.

Photoreactions and Structural Changes of Anabaena Sensory Rhodopsin

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria. The gene encoding ASR forms a single operon with ASRT (ASR transducer) which is a 14 kDa soluble protein, suggesting that ASR functions as a photochromic sensor by activating the soluble transducer. This article reviews the detailed photoreaction processes of ASR, which were studied by low-temperature Fourier-transform infrared (FTIR) and UV-visible spectroscopy. The former research reveals that the retinal isomerization is similar to bacteriorhodopsin (BR), but the hydrogen-bonding network around the Schiff base and cytoplasmic region is different. The latter study shows the stable photoproduct of the all-trans form is 100% 13-cis, and that of the 13-cis form is 100% all-trans. These results suggest that the structural changes of ASR in the cytoplasmic domain play important roles in the activation of the transducer protein, and photochromic reaction is optimized for its sensor function.

The Anabaena sensory rhodopsin transducer defines a novel superfamily of prokaryotic small-molecule binding domains

The Anabaena sensory rhodopsin transducer (ASRT) is a small protein that has been claimed to function as a signaling molecule downstream of the cyanobacterial sensory rhodopsin. However, orthologs of ASRT have been detected in several bacteria that lack rhodopsin, raising questions about the generality of this function. Using sequence profile searches we show that ASRT defines a novel superfamily of β-sandwich fold domains. Through contextual inference based on domain architectures and predicted operons and structural analysis we present strong evidence that these domains bind small molecules, most probably sugars. We propose that the intracellular versions like ASRT probably participate as sensors that regulate a diverse range of sugar metabolism operons or even the light sensory behavior in Anabaena by binding sugars or related metabolites. We also show that one of the extracellular versions define a predicted sugar-binding structure in a novel cell-surface lipoprotein found across actinobacteria, including several pathogens such as Tropheryma, Actinomyces and Thermobifida. The analysis of this superfamily also provides new data to investigate the evolution of carbohydrate binding modes in β-sandwich domains with very different topologies.Reviewers: This article was reviewed by M. Madan Babu and Mark A. Ragan.

FTIR Study of the L Intermediate of Anabaena Sensory Rhodopsin: Structural Changes in the Cytoplasmic Region

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria. The gene encoding ASR forms a single operon with ASRT (ASR transducer) that is a 14 kDa soluble protein, suggesting that ASR functions as a photochromic sensor by activating the soluble transducer. One of the characteristics of ASR is that the formation of the M intermediate accompanies a proton transfer from the Schiff base to Asp217 in the cytoplasmic side [Shi, L., Yoon, S. R., Bezerra, A. G., Jr., Jung, K. H., and Brown, L. S. (2006) J. Mol. Biol. 358, 686-700], in remarkable contrast to other archaeal-type rhodopsins such as a light-driven proton-pump, bacteriorhodopsin (BR). In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the all- trans form of ASR at 170 K, and compared the structural changes in the L intermediate with those of BR. The ASR L minus ASR difference spectra were essentially similar to those for BR, suggesting common structures for the L state in ASR and BR. On the other hand, unique CO stretching bands of a protonated carboxylic acid were observed at 1722 (+) and 1703 (-) cm (-1) at pH 5 and 7, and assigned to Glu36 by use of mutants. Glu36 is located at the cytoplasmic side, and the distance from the Schiff base is about 20 A. This result shows the structural changes at the cytoplasmic surface in ASR L. pH-dependent frequency change was also observed for a water stretching vibration, suggesting that the water molecule is involved in a hydrogen-bonding network with Glu36 and Asp217. Unique hydrogen-bonding network in the cytoplasmic domain of ASR will be discussed.

Crystal Structure of the Anabaena Sensory Rhodopsin Transducer

We present crystal structures of the Anabaena sensory rhodopsin transducer ( ASRT), a soluble cytoplasmic protein that interacts with the first structurally characterized eubacterial retinylidene photoreceptor Anabaena sensory rhodopsin ( ASR). Four crystal structures of ASRT from three different spacegroups were obtained, in all of which ASRT is present as a planar ( C4) tetramer, consistent with our characterization of ASRT as a tetramer in solution. The ASRT tetramer is tightly packed, with large interfaces where the well-structured β-sandwich portion of the monomers provides the bulk of the tetramer-forming interactions, and forms a flat, stable surface on one side of the tetramer (the β-face). Only one of our four different ASRT crystals reveals a C-terminal α-helix in the otherwise all-β protein, together with a large loop from each monomer on the opposite face of the tetramer (the α-face), which is flexible and largely disordered in the other three crystal forms. Gel-filtration chromatography demonstrated that ASRT forms stable tetramers in solution and isothermal microcalorimetry showed that the ASRT tetramer binds to ASR with a stoichiometry of one ASRT tetramer per one ASR photoreceptor with a K d of 8 μM in the highest affinity measurements. Possible mechanisms for the interaction of this transducer tetramer with the ASR photoreceptor via its flexible α-face to mediate transduction of the light signal are discussed.

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.