Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin.

Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin, which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of Na+ pump rhodopsin from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.

Synthesis and biological evaluation of nectriatide derivatives, potentiators of amphotericin B activity.

Nectriatide 1a, a naturally occurring cyclic tetrapeptide, has been reported to a potentiator of amphotericin B (AmB) activity. In order to elucidate its structure-activity relationships, we synthesized nectriatide derivatives with different amino acids in solution-phase synthesis and evaluated AmB-potentiating activity against Candida albicans. Among them, C-and N-terminal protected linear peptides were found to show the most potent AmB-potentiating activity.

Anti-fatigue activity of methyl dihydrojasmonate and linalool in a rat model evaluated by a novel index for neuro-immune and oxidative stress interactions.

Avoiding fatigue is a long-standing challenge in both healthy and diseased individuals. Establishing objective standard markers of fatigue is essential to evaluate conditions in spatiotemporally different locations and individuals and identify agents to fight against fatigue. Herein, we introduced a novel method for evaluating fatigue using nervous system markers (including dopamine, adrenaline, and noradrenaline), various cytokine levels (such as interleukin [IL]-1ß, tumor necrosis factor [TNF]-α, IL-10, IL-2, IL-5 and IL-17A), and oxidative stress markers (such as diacron-reactive oxygen metabolites [d-ROMs] and biological antioxidant potential [BAP]) in a rat fatigue model. Using this method, the anti-fatigue effects of methyl dihydrojasmonate (MDJ) and linalool, the fragrance/flavor compounds used in various products, were assessed. Our method evaluated the anti-fatigue effects of the aforementioned compounds based on the changes in levels of the nerves system markers, cytokines, and oxidative stress markers. MDJ exerted more potent anti-fatigue effects than linalool. In conclusion, the reported method could serve as a useful tool for fatigue studies and these compounds may act as effective therapeutic agents for abrogating fatigue symptoms.

Design and Synthesis of Monobody Variants with Low Immunogenicity.

Mirror-image proteins (d-proteins) are promising scaffolds for drug discovery because of their high proteolytic stability and low immunogenic properties. Facile and reproducible processes for the preparation of functional d-proteins are required for their application in therapeutic biologics. In this study, we designed and synthesized a novel monobody variant with two cysteine substitutions that facilitate the synthetic process via sequential native chemical ligations and improve protein stability by disulfide bond formation. The synthetic anti-GFP monobody in this model study exhibited good binding affinity to the target enhanced green fluorescent protein. In vivo administration of the synthetic anti-GFP monobody (l-monobody) to mice induced antidrug antibody (ADA) production, whereas no ADA production was observed following immunization with the mirror-image anti-GFP monobody (d-monobody). These results suggest that the synthetic d-monobody is a non-antibody protein scaffold with low immunogenic properties.

Identification of anti-SARS-CoV-2 agents based on flavor/fragrance compositions that inhibit the interaction between the virus receptor binding domain and human angiotensin converting enzyme 2.

Coronavirus disease 2019 (COVID-19) pandemic poses a threat to human beings and numerous cases of infection as well as millions of victims have been reported. The binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor binding domain (RBD) to human angiotensin converting enzyme 2 (hACE2) is known to promote the engulfment of the virus by host cells. Employment of flavor/fragrance compositions to prevent SARS-CoV-2 infection by inhibiting the binding of viral RBD (vRBD) to hACE2 might serve as a favorable, simple, and easy method for inexpensively preventing COVID-19, as flavor/fragrance compositions are known to directly interact with the mucosa in the respiratory and digestive systems and have a long history of use and safety assessment. Herein we report the results of screening of flavor/fragrance compositions that inhibit the binding of vRBD to hACE2. We found that the inhibitory effect was observed with not only the conventional vRBD, but also variant vRBDs, such as L452R, E484K, and N501Y single-residue variants, and the K417N+E484K+N501Y triple-residue variant. Most of the examined flavor/fragrance compositions are not known to have anti-viral effects. Cinnamyl alcohol and Helional inhibited the binding of vRBD to VeroE6 cells, a monkey kidney cell line expressing ACE2. We termed the composition with inhibitory effect on vRBD-hACE2 binding as "the molecularly targeted flavor/fragrance compositions". COVID-19 development could be prevented by using these compositions with reasonable administration methods such as inhalation, oral administration, and epidermal application.