Biophysics and physicobiology最新文献

Heliorhodopsin (HeR) is a family of microbial rhodopsin discovered in 2018, whose genes are found from archaea, bacteria, unicellular eukaryotes, and giant viruses. Viral heliorhodopsins are classified into VHeR1-4 based on their amino acid sequences, and we previously reported the proton transport activity for V2HeR3. In this study, we report molecular properties of V2HeR2. V2HeR2 contains all-trans retinal predominantly in the dark, and the protonated Schiff base is stabilized by a counterion. The photocycle is described by the sequentially-formed K, M, and O intermediates. The O intermediate with a long lifetime (15.8 sec) and negligible ion transport activity implicate the light sensor function for V2HeR2, which is also the case for many HeRs. FTIR spectroscopy revealed that the chromophore structure is a distorted 13-cis form in the K and O intermediates. Although these properties are common for other HeRs, FTIR spectroscopy gain unique structural factors in the active O intermediate. The 13-cis chromophore is highly distorted near the Schiff base, and the hydrogen bond of the Schiff base is weaker than the resting state. The long-lived O intermediate with the distorted 13-cis retinal and without hydrogen bond of the Schiff base is unique in V2HeR2, which is regulated by the surrounding protein moiety. Strengthened hydrogen bond in amide-I band in the O intermediate of V2HeR2 is opposite to the case in Thermoplasmatales archaeon HeR (TaHeR) and HeR 48C12. Unique protein structural changes in V2HeR2, TaHeR, and HeR 48C12 are possibly correlated to different interaction with their partner proteins.

Finding early trigger genes involved in cell-fate-determining processes is important for understanding molecular mechanisms of, e.g., differentiation and disease progression. One of the powerful tools for the finding is hypothesis-free omics measurements, e.g., gene expression analysis (transcriptome analysis) by RNA sequencing (RNA-seq). However, because whole single-cell RNA-seq requires cell disruption and the fate of the disrupted cell is generally unknown, it is difficult to find fate-related genes by single-cell RNA-seq profiles, especially in the early stages of cell-fate determination. Meanwhile, deep learning has successfully predicted cell fates using individual cell images. Here, we developed an approach by integrating image-based cell-fate prediction using deep learning and single-cell whole-transcriptome analysis to find differentially expressed genes (DEGs) between different predicted fates. As a proof of principle, we applied this approach to cells fated to die and survive. First, we applied temporary heat stress to a mammalian cell line to induce a certain fraction of cells to die, and performed time-lapse imaging to observe this process. Second, we made image-based deep learning models trained with our dataset for the cell fate prediction (survival and death). Third, we picked the cells after another time-lapse imaging and performed single-cell RNA-seq. Finally, we compared the transcriptomes between cells predicted to die and survive. We successfully detected the DEGs when the transcriptomic profiles did not show clear multiple clusters that may correspond to the heat-induced different fates in a dimension-reduced plane. Our approach may contribute to a deeper understanding of cell-fate regulation and new molecular marker detection.

The bacterial peptidoglycan layer plays an important role in protecting the bacteria from turgor pressure, viruses, and predators. However, it also acts as a barrier in transmitting forces generated on the cell membrane to adhesion proteins on the surface during gliding locomotion. In this study, peptidoglycan layers were isolated from two species of gliding diderm, i.e., gram-negative bacteria, and their structures were visualized by quick-freeze deep-etch replica electron microscopy. The horizontal bonding of peptidoglycan did not differ obviously among the three species. However, the diameter of pores in the peptidoglycan layer of M. xanthus and the area of surface pores were 51 nm and 14.6%, respectively, which were significantly larger than those of E. coli (32 nm and 5.8%) and F. johnsoniae (29 nm and 7.0%). Based on this, we discussed the mechanism by which diderm bacteria transmit forces across the PG layer to the bacterial surface.

The crystal structure of Rubrobacter xylanophilus rhodopsin (RxR) reveals a triangular cluster of three water molecules (W413, W415, and W419) at the extracellular proton-release site, near Glu187 and Glu197. Using a quantum mechanical/molecular mechanical approach, we identified the structural nature of this unique water cluster. The triangular shape is best reproduced when all three water molecules are neutral H₂O with protonated Glu187 and deprotonated Glu197. Attempts to place H₃O⁺ at any of these water molecules result in spontaneous proton transfer to one of the acidic residues and significant distortion from the crystal structure. The plane defined by the triangular water cluster extends into the guanidinium plane of Arg71, with both aligned along the W413...W419 axis. This extended plane lies nearly perpendicular to a five-membered, ring-like H-bond network involving two carboxyl oxygen atoms from Glu187 and one from Glu197. The resulting bipartite planar architecture, defined by the water triangle, Arg71, and the Glu187/Glu197 network may reflect the exceptional thermal stability in RxR.

This study aimed to elucidate the impact of polyethylene glycol (PEG) conjugation on protein-protein interactions by investigating the properties of PEG-conjugated actin (PEG-actin). Various PEG molecules were covalently bound to actin monomers by reacting maleimide groups with Cys374 on the actin. The apparent polymerization rate constant (k_on ^app ) and the critical concentration (Cc) were measured by fluorescence spectroscopy using pyrene-labeled actin, as a function of the portion of PEG-actin and the molecular mass of the conjugated PEG (750 to 10000 Da). The k_on ^app gradually decreased as the percentage of PEG-actin increased. At 90% PEG-actin, the k_on ^app decreased substantially as the PEG size increased, resulting from a modulation of C-terminus by the conjugated PEGs and their steric hindrance. The Cc was slightly increased by PEG conjugation in the content by up to 50%. Meanwhile, 90% PEG-actin exhibited a substantial increase in Cc. The Cc was almost linearly related to the gyration radius of PEG. These results suggest that the PEG conjugation to actin impedes the association of actin with the filament in a PEG size-dependent manner. Furthermore, the stability of PEG-actin against an extrinsic factor was assessed. PEG-actins >2000 Da were more susceptible to digestion than intact actin when the PEG-actin monomer was subjected to α-chymotrypsin. Thus, conjugation of PEG to Cys374 on actin did not protect actin monomers against their proteolysis by α-chymotrypsin.

Quality assessment and characterization of liquid milk by means of electronic sensor remains an intensive area of research. In this work, cylinder-in-cylinder type sample holder is fabricated to measure the bioimpedance of milk as a function of frequency. Experiments on bioimpedance spectroscopy were conducted during adulteration (at control temperature, 23°C) of branded (pasteurized) milk as well as raw milk. Cole equivalent circuit is considered as a characterizing model for milk. Cole parameters were extracted from the experimental data. From Cole parameters, relaxation-time was estimated and state of the milk sample has been expressed in terms of relaxation-time. Analysis of variance was performed on relaxation-time of the samples to gain statistical significance. Our method is capable to discriminate liquid milk from different commercial brands. It was found that the relaxation-time decreases monotonically with the progression of adulteration time for all kinds of milk considered in this study. From the changes in relaxation-time, the adulteration was found to be significant in the first three hours. Hence it is not advisable to consume milk after two hours of adulteration if kept at 23°C. Dominant biochemical pathways responsible for adulteration of milk are also presented.

Predicting the binding affinity between proteins and ligands is a critical task in drug discovery. Although various computational methods have been proposed to estimate ligand target affinity, the method of Yasuda et al. (2022) ranks affinities based on the dynamic behavior obtained from molecular dynamics (MD) simulations without requiring structural similarity among ligand substituents. Thus, its applicability is broader than that of relative binding free energy calculations. However, their approach suffers from high computational costs due to the extensive simulation time and the deep learning computations needed for each ligand pair. Moreover, in the absence of experimental ΔG values (oracle), the sign of the correlation can be misinterpreted. In this study, we present an alternative approach inspired by Yasuda et al.'s method, offering an alternative perspective by replacing the distance metric and reducing computational cost. Our contributions are threefold: (1) By introducing the Jensen-Shannon (JS) divergence, we eliminate the need for deep learning-based similarity estimation, thereby significantly reducing computation time; (2) We demonstrate that production run simulation times can be halved while maintaining comparable accuracy; and (3) We propose a method to predict the sign of the correlation between the first principal component (PC1) and ΔG by using coarse ΔG estimations obtained via AutoDock Vina.

Chrimson is cation-conducting channelrhodopsin (CCR) with the most red-shifted absorption spectrum, rendering itself as one of the most promising optogenetic tools. However, the molecular mechanisms underlying its red-shifted absorption have not been completely clarified yet. Here, we found a CCR gene showing high sequence similarity to Chrimson from Lake Hula through freshwater metatranscriptome sampling. Interestingly, despite its high similarity to Chrimson, this CCR-named HulaChrimson-showed significantly blue-shifted action and absorption spectra compared to those of Chrimson. Mutations of amino acid residues, which are prominently different from those in Chrimson, in HulaChrimson did not reproduce the red-shifted absorption of Chrimson, suggesting the color-tuning between these proteins achieved by organizing the entire protein architecture, particularly in the broad hydrogen bonding network around the retinal Schiff base counterion, rather than by the difference in several specific residues. The optical characteristics of HulaChrimson distinct from those of Chrimson provide a basis for understanding the color-tuning mechanisms of channelrhodopsins.