Bacteriophages targeting Enterococcus faecalis enhance the therapeutic efficacy of levodopa in an MPTP-induced Parkinson's disease mouse model with E. faecalis gut colonization.

Despite the intensive research on gut microbiome-associated diseases over the past 20 years, pharmacological methods for effectively eliminating pathobionts remain unsatisfactory. This study investigated the therapeutic potential of bacteriophages against Enterococcus faecalis, in which bacterial tyrosine decarboxylase (TDC) converts orally administered levodopa (L-DOPA) to dopamine, in an MPTP mouse model of Parkinson's disease (PD). E. faecalis bacteriophages PBEF62, PBEF66, and PBEF67 (4 × 1010 PFU total/200 µl/day), and E. faecalis cells (2 × 109 CFU/200 µl/day) were orally administered at 2-h intervals before every MPTP (i.p.) and/or L-DOPA (p.o.) treatments for 13 days. The relative abundances of E. faecalis cells and bacteriophages in the feces peaked at 4 and 12 h after administration and gradually decreased by 12 and 48 h, respectively. While the administration of E. faecalis cells eliminated the beneficial effect of L-DOPA on MPTP-induced behavioral deficits, as assessed by cylinder and rotarod tests, the co-administration of bacteriophages with bacterial cells restored this effect. The modulating effects of L-DOPA, E. faecalis, and bacteriophages on PD behavior were closely associated with choline acetyltransferase expression levels in the striatum but not with tyrosine hydroxylase in the substantia nigra of each group. Recurrence and extinction of PD behaviors following treatment with E. faecalis and/or bacteriophages were also coincident with the dopamine levels in the blood and brain tissues of PD mice. The effectiveness of L-DOPA was restored after the three types of E. faecalis bacteriophages selectively eliminated E. faecalis cells, along with the TDC gene copies and transcripts responsible for converting L-DOPA to dopamine in the gastrointestinal tract. In conclusion, a combination of bacteriophages PBEF62, PBEF66, and PBEF67 targeting E. faecalis demonstrates potential as a valuable supplement to L-DOPA therapy for PD.

Clinical validation of artificial intelligence-based preoperative virtual reduction for Neer 3- or 4-part proximal humerus fractures.

BACKGROUND: If reduction images of fractures can be provided in advance with artificial-intelligence (AI)-based technology, it can assist with preoperative surgical planning. Recently, we developed the AI-based preoperative virtual reduction model for orthopedic trauma, which can provide an automatic segmentation and reduction of fractured fragments. The purpose of this study was to validate a quality of reduction model of Neer 3- or 4-part proximal humerus fractures established by AI-based technology. METHODS: To develop the AI-based preoperative virtual reduction model, deep learning performed the segmentation of fracture fragments, and a Monte Carlo simulation completed the virtual reduction to determine the best model. A total of 20 pre/postoperative three-dimensional computed tomography (CT) scans of proximal humerus fracture were prepared. The preoperative CT scans were employed as the input of AI-based automated reduction (AI-R) to deduce the reduction models of fracture fragments, meanwhile, the manual reduction (MR) was conducted using the same CT images. Dice similarity coefficient (DSC) and intersection over union (IoU) between the reduction model from the AI-R/MR and postoperative CT scans were evaluated. Working times were compared between the two groups. Clinical validity agreement (CVA) and reduction quality score (RQS) were investigated for clinical validation outcomes by 20 orthopedic surgeons. RESULTS: The mean DSC and IoU were better when using AI-R that when using MR (0.78 ± 0.13 vs. 0.69 ± 0.16, p < 0.001 and 0.65 ± 0.16 vs. 0.55 ± 0.18, p < 0.001, respectively). The working time of AI-R was, on average, 1.41% of that of MR. The mean CVA of all cases was 81%±14.7% (AI-R, 82.25%±14.27%; MR, 76.75%±14.17%, p = 0.06). The mean RQS was significantly higher when AI-R compared with MR was used (91.47 ± 1.12 vs. 89.30 ± 1.62, p = 0.045). CONCLUSION: The AI-based preoperative virtual reduction model showed good performance in the reduction model in proximal humerus fractures with faster working times. Beyond diagnosis, classification, and outcome prediction, the AI-based technology can change the paradigm of preoperative surgical planning in orthopedic surgery. LEVEL OF EVIDENCE: Level IV.

Proton-pumping photoreceptor controls expression of ABC transporter by regulating transcription factor through light.

Light is a significant factor for living organisms with photosystems, like microbial rhodopsin-a retinal protein that functions as an ion pump, channel, and sensory transduction. Gloeobacter violaceus PCC7421, has a proton-pumping rhodopsin gene, the Gloeobacter rhodopsin (GR). The helix-turn-helix family of transcriptional regulators has various motifs, and they regulate gene expression in the presence of various metal ions. Here, we report that active proton outward pumping rhodopsin interacted with the helix-turn-helix transcription regulator and regulated gene expression. This interaction is confirmed using ITC analysis (KD of 8 µM) and determined the charged residues required. During in vitro experiments using fluorescent and luciferase reporter systems, ATP-binding cassette (ABC) transporters and the self-regulation of G. violaceus transcriptional regulator (GvTcR) are regulated by light, and gene regulation is observed in G. violaceus using the real-time polymerase chain reaction. These results expand our understanding of the natural potential and limitations of microbial rhodopsin function.

Unveiling the critical role of K+ for xanthorhodopsin expression in E. coli.

Xanthorhodopsin (XR), a retinal-binding 7-transmembrane protein isolated from the eubacterium Salinibacter ruber, utilizes two chromophores (retinal and salinixanthin (SAL)) as an outward proton pump and energy-donating carotenoid. However, research on XR has been impeded owing to limitations in achieving heterogeneous expression of stable forms and high production levels of both wild-type and mutants. We successfully expressed wild-type and mutant XRs in Escherichia coli in the presence of K+. Achieving XR expression requires significant K+ and a low inducer concentration. In particular, we highlight the significance of Ser-159 in helix E located near Gly-156 (a carotenoid-binding position) as a critical site for XR expression. Our findings indicate that replacing Ser-159 with a smaller amino acid, alanine, can enhance XR expression in a manner comparable to K+, implying that Ser-159 poses a steric hindrance for pigment formation in XR. In the presence of K+, the proton pumping and photocycle of the wild-type and mutants were characterized and compared; the wild-type result suggests similar properties to the first reported XR isolation from the S. ruber membrane fraction. We propose that the K+ gradient across the cell membrane of S. ruber serves to uphold the membrane potential of the organism and plays a role in the expression of proteins, such as XR, as demonstrated in our study. Our findings deepen the understanding of adaptive protein expression, particularly in halophilic organisms. We highlight salt selection as a promising strategy for improving protein yield and functionality.

Heliorhodopsin-mediated light-modulation of ABC transporter.

Heliorhodopsins (HeRs) have been hypothesized to have widespread functions. Recently, the functions for few HeRs have been revealed; however, the hypothetical functions remain largely unknown. Herein, we investigate light-modulation of heterodimeric multidrug resistance ATP-binding cassette transporters (OmrDE) mediated by Omithinimicrobium cerasi HeR. In this study, we classifiy genes flanking the HeR-encoding genes and identify highly conservative residues for protein-protein interactions. Our results reveal that the interaction between OcHeR and OmrDE shows positive cooperatively sequential binding through thermodynamic parameters. Moreover, light-induced OcHeR upregulates OmrDE drug transportation. Hence, the binding may be crucial to drug resistance in O. cerasi as it survives in a drug-containing habitat. Overall, we unveil a function of HeR as regulatory rhodopsin for multidrug resistance. Our findings suggest potential applications in optogenetic technology.

Real-time Visualization of Transcribed mRNA via Click Chemistry in a Liposomal Space.

We present a CuAAC (Copper-Catalyzed Azide-Alkyne Cycloaddition) reaction protocol designed for the visualization of mRNA. To achieve this, we synthesized stable mRNA molecules incorporating the modified nucleoside analog, EU, a crucial element for fluorophore attachment. Leveraging this modified mRNA, we successfully executed the CuAAC reaction, wherein the pro-fluorophore, coumarin, was conjugated to EU on the mRNA through our meticulously designed CuAAC process. This innovative approach resulted in the emission of fluorescence, enabling both precise quantification and visual observation of mRNA. Furthermore, we demonstrated the feasibility of concurrent mRNA synthesis and visualization by seamlessly integrating the CuAAC reaction mix into the mRNA transcription process. Additionally, our novel methodology opens avenues for prospective real-time monitoring of mRNA transcription within artificial cells. These advancements hold significant promise for expanding our comprehension of fundamental cellular processes and finding applications across diverse biological contexts in the future.

Impact of protein-chromophore interaction on the retinal excited state and photocycle of Gloeobacter rhodopsin: role of conserved tryptophan residues.

The function of microbial as well as mammalian retinal proteins (aka rhodopsins) is associated with a photocycle initiated by light excitation of the retinal chromophore of the protein, covalently bound through a protonated Schiff base linkage. Although electrostatics controls chemical reactions of many organic molecules, attempt to understand its role in controlling excited state reactivity of rhodopsins and, thereby, their photocycle is scarce. Here, we investigate the effect of highly conserved tryptophan residues, between which the all-trans retinal chromophore of the protein is sandwiched in microbial rhodopsins, on the charge distribution along the retinal excited state, quantum yield and nature of the light-induced photocycle and absorption properties of Gloeobacter rhodopsin (GR). Replacement of these tryptophan residues by non-aromatic leucine (W222L and W122L) or phenylalanine (W222F) does not significantly affect the absorption maximum of the protein, while all the mutants showed higher sensitivity to photobleaching, compared to wild-type GR. Flash photolysis studies revealed lower quantum yield of trans-cis photoisomerization in W222L as well as W222F mutants relative to wild-type. The photocycle kinetics are also controlled by these tryptophan residues, resulting in altered accumulation and lifetime of the intermediates in the W222L and W222F mutants. We propose that protein-retinal interactions facilitated by conserved tryptophan residues are crucial for achieving high quantum yield of the light-induced retinal isomerization, and affect the thermal retinal re-isomerization to the resting state.

AT-specific DNA visualization revisits the directionality of bacteriophage λ DNA ejection.

In this study, we specifically visualized DNA molecules at their AT base pairs after in vitro phage ejection. Our AT-specific visualization revealed that either end of the DNA molecule could be ejected first with a nearly 50% probability. This observation challenges the generally accepted theory of Last In First Out (LIFO), which states that the end of the phage λ DNA that enters the capsid last during phage packaging is the first to be ejected, and that both ends of the DNA are unable to move within the extremely condensed phage capsid. To support our observations, we conducted computer simulations that revealed that both ends of the DNA molecule are randomized, resulting in the observed near 50% probability. Additionally, we found that the length of the ejected DNA by LIFO was consistently longer than that by First In First Out (FIFO) during in vitro phage ejection. Our simulations attributed this difference in length to the stiffness difference of the remaining DNA within the phage capsid. In conclusion, this study demonstrates that a DNA molecule within an extremely dense phage capsid exhibits a degree of mobility, allowing it to switch ends during ejection.

A Bibliometric Analysis of the Field of Computer-Assisted Orthopedic Surgery during 2002-2021.

Background: This study aimed to investigate the characteristics of research articles and research trends in computer-assisted orthopedic surgery (CAOS) by conducting bibliometric analyses. Methods: CAOS-related research articles published in international journals from 2002 to 2021 were collected using the PubMed database and analyzed using the bibliometric method. Their publication year, journal name, corresponding author's country name, and the number of citations of all collected articles were noted. Contents of the articles were analyzed to evaluate the time point and anatomical site at which the digital technique was applied. Further, the 20-year period was divided into two halves of 10 years each to analyze the research trends. Results: A total of 639 CAOS-related articles were identified. An average of 32.0 CAOS-related articles were published annually, with an average of 20.6 and 43.3 published in the first half and second half, respectively. Of all articles, 47.6% were published in the top 10 journals, and 81.2% were written in the top 10 countries. The total numbers of citations were 11.7 and 6.3 in the first and second halves, respectively, but the average annual number of citations was higher in the second half than in the first one. Articles on application of digital techniques during surgery were 62.3% and those on pre-surgery application were 36.9%. Further, articles in the knee (39.0%), spine (28.5%), and hip and pelvis (21.5%) fields accounted for 89.0% of the total publications. But the increase in publications in the said period was highest in the fields of the hand and wrist (+1,300.0%), ankle (+466.7%), and shoulder (+366.7%). Conclusions: Over the last 20 years, the publication of CAOS-related research articles in international journals has grown steadily. Although the knee, spine, hip, and pelvis fields account for most CAOS-related research, research in new fields is also increasing. This study analyzed the types of articles and trends in CAOS-related research and provided useful information for future research in the field of CAOS.

Surgery-related anxiety on geriatric patients undergoing total knee arthroplasty: a retrospective observational study.

BACKGROUND: The prevalence of anxiety in patients undergoing total knee arthroplasty (TKA) and its association with postoperative functions are well known; however, the levels of anxiety or anxiety-related characteristics are unknown. This study aimed to investigate the prevalence of clinically significant state anxiety in geriatric patients undergoing TKA for osteoarthritis (OA) of the knee and to evaluate the anxiety-related characteristics experienced by these patients pre- and post-operatively. METHODS: This retrospective observational study recruited patients who had undergone TKA for knee OA using general anesthesia between February 2020 and August 2021. The study participants were geriatric patients older than 65 years who had moderate or severe OA. We evaluated patient characteristics including age, sex, body mass index, smoking status, hypertension, diabetes, and cancer. We assessed their levels of anxiety status using the STAI-X which comprises 20-item scales. Clinically meaningful state anxiety was defined as a total score of 52 or higher. An independent Student's t-test was used to determine differences of STAI score between subgroups in terms of patient characteristics. And patients were asked to complete questionnaires, which assessed four areas: (1) the main cause of anxiety; (2) the most helpful factor in overcoming anxiety before surgery; (3) the most helpful factor in reducing anxiety after surgery; and (4) the most anxious moment during the entire process. RESULTS: The mean STAI score of patients who underwent TKA was 43.0 points and 16.4% of patients experienced clinically significant state anxiety. The current smoking status affect STAI score and the proportion of patients with clinically meaningful state anxiety. The most common cause of preoperative anxiety was the surgery itself. Overall, 38% of patients reported that they experienced the greatest level of anxiety when the surgeon had recommended TKA in the outpatient clinic. The trust in the medical staff before surgery and the surgeon's explanations after surgery helped the most in reducing anxiety. CONCLUSIONS: One in six patients before TKA experience clinically meaningful state anxiety, and about 40% of patients experience anxiety from the time they are recommended for surgery. Patients tended to overcome anxiety before TKA through trust in the medical staff, and the surgeon's explanations after surgery was found to be helpful in reducing anxiety.

Retinal-Carotenoid Interactions in a Sodium-Ion-Pumping Rhodopsin: Implications on Oligomerization and Thermal Stability.

Microbial rhodopsin (also called retinal protein)-carotenoid conjugates represent a unique class of light-harvesting (LH) complexes, but their specific interactions and LH properties are not completely elucidated as only few rhodopsins are known to bind carotenoids. Here, we report a natural sodium-ion (Na+)-pumping Nonlabens (Donghaeana) dokdonensis rhodopsin (DDR2) binding with a carotenoid salinixanthin (Sal) to form a thermally stable rhodopsin-carotenoid complex. Different spectroscopic studies were employed to monitor the retinal-carotenoid interaction as well as the thermal stability of the protein, while size-exclusion chromatography (SEC) and homology modeling are performed to understand the protein oligomerization process. In analogy with that of another Na+-pumping protein Krokinobacter eikastus rhodopsin 2 (KR2), we propose that DDR2 (studied concentration range: 2 × 10-6 to 4 × 10-5 M) remains mainly as a pentamer at room temperature and neutral pH, while heating above 55 °C partially converted it into a thermally less stable oligomeric form of the protein. This process is affected by both the pH and concentration. At high concentrations (4 × 10-5 to 2 × 10-4 M), the protein adopts a pentamer form reflected in the excitonic circular dichroism (CD) spectrum. In the presence of Sal, the thermal stability of DDR2 is increased significantly, and the pigment is stable even at 85 °C. The results presented could have implications in designing stable rhodopsin-carotenoid antenna complexes.

Heliorhodopsin binds and regulates glutamine synthetase activity.

Photoreceptors are light-sensitive proteins found in various organisms that respond to light and relay signals into the cells. Heliorhodopsin, a retinal-binding membrane protein, has been recently discovered, however its function remains unknown. Herein, we investigated the relationship between Actinobacteria bacterium IMCC26103 heliorhodopsin (AbHeR) and an adjacent glutamine synthetase (AbGS) in the same operon. We demonstrate that AbHeR binds to AbGS and regulates AbGS activity. More specifically, the dissociation constant (Kd) value of the binding between AbHeR and AbGS is 6.06 µM. Moreover, the absence of positively charged residues within the intracellular loop of AbHeR impacted Kd value as they serve as critical binding sites for AbGS. We also confirm that AbHeR up-regulates the biosynthetic enzyme activity of AbGS both in vitro and in vivo in the presence of light. GS is a key enzyme involved in nitrogen assimilation that catalyzes the conversion of glutamate and ammonia to glutamine. Hence, the interaction between AbHeR and AbGS may be critical for nitrogen assimilation in Actinobacteria bacterium IMCC26103 as it survives in low-nutrient environments. Overall, the findings of our study describe, for the first time, to the best of our knowledge, a novel function of heliorhodopsin as a regulatory rhodopsin with the capacity to bind and regulate enzyme activity required for nitrogen assimilation.

Heliorhodopsin Helps Photolyase to Enhance the DNA Repair Capacity.

Light quality is a significant factor for living organisms that have photosensory systems, such as rhodopsin, a seven alpha-helical transmembrane protein with the retinal chromophore. Here, we report, for the first time, the function of new rhodopsin, which is an inverted 7-transmembrane protein, isolated from Trichococcus flocculiformis. T. flocculiformis heliorhodopsin (TfHeR) works as a regulatory helper rhodopsin that binds with class 2 cyclobutane pyrimidine dimer (CPDII) photolyase to broaden the spectrum and upregulate DNA repair activity. We have confirmed their interaction through isothermal titration calorimetry (dissociation constant of 21.7 µM) and identified the charged residues for the interaction. Based on in vivo and in vitro experiments, we showed that the binding of heliorhodopsin with photolyase improved photolyase activity by about 3-fold to repair UV-caused DNA damage. Also, the DNA repair activity of TfHeR/T. flocculiformis photolyase (TfPHR) was observed in the presence of green light. Our results suggested that heliorhodopsin directly controls the activity of photolyase and coevolves to broaden the activity spectrum by protein-protein interaction. IMPORTANCE This study reports a function for Heliorhodopsin working as a regulatory helper rhodopsin that with CPDII photolyase to broaden the spectrum and upregulating the DNA repair activity. Our results suggested that heliorhodopsin directly controls photolyase activity and coevolves to broaden the DNA repair capacity by protein-protein interaction.

Characterization of the Interfacial Structures of Core/Shell CdSe/ZnS QDs.

Core/shell quantum dots (QDs) have been extensively studied, yet their optical properties widely vary among studies. Such variation may arise from the variation in interfacial structures induced by the subtle difference in each synthetic procedure. Here, we studied the interfacial structures of CdSe/ZnS QDs using the time-of-flight medium energy ion-scattering spectroscopy (TOF-MEIS), which offers the radial elemental distributions as well as the overall elemental compositions of QDs. The TOF-MEIS spectra provided strong evidence for the existence of an alloyed layer at the interface between CdSe and ZnS in typical CdSe/ZnS QDs. On the basis of the emission and absorption spectra of QDs sampled during the synthesis, we conclude that such interfacial alloying is caused by the dissolution of CdSe seeds during the synthesis steps. Such a dissolution mechanism is further corroborated by the observation that the ligand environment of solvent (X or L type) leads to different shapes of interfaces.

Fabrication of a Tailored, Hybrid Extracellular Matrix Composite.

The extracellular matrix (ECM) is a network of connective fibers that supports cells living in their surroundings. Native ECM, generated by the secretory products of each tissue's resident cells, has a unique architecture with different protein composition depending on the tissue. Therefore, it is very difficult to artificially design in vivo architecture in tissue engineering. In this study, a hybrid ECM scaffold from the basic structure of fibroblast-derived cellular ECMs is fabricated by adding major ECM components of fibronectin (FN) and collagen (COL I) externally. It is confirmed that while maintaining the basic structure of the native ECM, major protein components can be regulated. Then, decellularization is performed to prepare hybrid ECM scaffolds with various protein compositions and it is demonstrated that a liver-mimicking fibronectin (FN)-rich hybrid ECM promoted successful settling of H4IIE rat hepatoma cells. The authors believe that their method holds promise for the fabrication of scaffolds that provide a tailored cellular microenvironment for specific organs and serve as novel pathways for the replacement or regeneration of specific organ tissues.

Carotenoid binding in Gloeobacteria rhodopsin provides insights into divergent evolution of xanthorhodopsin types.

The position of carotenoid in xanthorhodopsin has been elucidated. However, a challenging expression of this opsin and a complex biosynthesis carotenoid in the laboratory hold back the insightful study of this rhodopsin. Here, we demonstrated co-expression of the xanthorhodopsin type isolated from Gloeobacter violaceus PCC 7421-Gloeobacter rhodopsin (GR) with a biosynthesized keto-carotenoid (canthaxanthin) targeting the carotenoid binding site. Direct mutation-induced changes in carotenoid-rhodopsin interaction revealed three crucial features: (1) carotenoid locked motif (CLM), (2) carotenoid aligned motif (CAM), and color tuning serines (CTS). Our single mutation results at 178 position (G178W) confirmed inhibition of carotenoid binding; however, the mutants showed better stability and proton pumping, which was also observed in the case of carotenoid binding characteristics. These effects demonstrated an adaptation of microbial rhodopsin that diverges from carotenoid harboring, along with expression in the dinoflagellate Pyrocystis lunula rhodopsin and the evolutionary substitution model. The study highlights a critical position of the carotenoid binding site, which significantly allows another protein engineering approach in the microbial rhodopsin family.

Does spiked tibial cement spacer reduce spacer-related problems in two-stage revision total knee arthroplasty for infection?

PURPOSE: Articulating cement spacers are frequently used in staged approaches for infected total knee arthroplasty (TKA). This study investigated whether a tibial cement spacer (TCS) with spikes could reduce spacer-related problems in two-stage revision TKA (R-TKA). METHODS: A total of 27 patients (27 knees; 10 men and 17 women) who underwent two-stage R-TKA for infected TKA were retrospectively analyzed. Group A comprised 12 patients who used TCS with spikes added to the bottom surface, whereas group B consisted of 15 patients who used conventional TCS with a flat bottom. For each group, plain radiographs were obtained after cement spacer implantation and before R-TKA to measure mediolateral (ML) translation and TCS's tilting angle. Patients' demographic data, ML translation of the TCS, and changes in the TCS's tilting angle between the groups were analyzed. RESULTS: The mean ML translation was significantly lower in group A than that in group B (1.7 mm vs. 5.4 mm, p = 0.04). The mean change in the tilting angle was significantly lower in group A than that in group B (4.5° vs. 19.4°, p = 0.047). CONCLUSION: The spiked TCS in two-stage R-TKA provides superior stability compared to the TCS with a conventional design.

Predictive Variables for Interventional Angiography among Patients with Knee Hemarthrosis.

Studies regarding the variables that could predict the success of conservative treatment for knee hemarthrosis are lacking. This retrospective study evaluated the laboratory variables of patients who had unsatisfactory results from conservative treatment for knee hemarthrosis. Twenty-nine patients conservatively treated for knee hemarthrosis were included and divided into two groups: group A comprised 14 patients who underwent interventional angiography and selective embolization due to failed conservative treatment, and group B comprised 15 patients with successful results after conservative treatment. The results of the serological and synovial fluid tests were evaluated. The mean number of synovial red blood cells (RBCs) was 1,905,857 cells/µL and 7730 cells/µL in groups A and B, respectively (p = 0.01), while the mean number of RBCs per high-power field (HPF) was 68.9 and 3.2, respectively (p < 0.01). Patients who underwent interventional angiography and selective embolization after failed conservative treatment for knee hemarthrosis had higher synovial RBC counts and RBC counts per HPF than those with successful outcomes after conservative treatment. It is necessary to carefully interpret the results of the synovial fluid analysis in patients with knee hemarthrosis; if the synovial fluid analysis shows a synovial RBC count greater than 81,500 and RBC count per HPF greater than 16.3, we recommend immediate interventional angiography rather than continuing conservative treatment.

Mutational analyses identify a single amino acid critical for colour tuning in proteorhodopsins.

Microbial rhodopsins are light-activated proteins that contain seven transmembrane alpha-helices. Spectral tuning in microbial rhodopsins is a useful optogenetic tool. In this study, we report a new site that controls spectral tuning. In the proteorhodopsins ISR34 and ISR36, a single amino-acid substitution at Cys189 caused an absorption maximum shift of 44 nm, indicating spectral tuning at a specific site. Comparison of single amino acid substitutions was conducted using photochemical and photobiological approaches. The maximum absorption for red-shift was measured for mutations at positions 189 and 105 in ISR34, both residues being equally important. Structural changes resulting from amino acid substitutions are related to pKa values, pumping activity and spectral tuning.

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.