Biophysics and physicobiology最新文献

Computerized molecular docking methodologies are pivotal in in-silico screening, a crucial facet of modern drug design. ChooseLD, a docking simulation software, combines structure- and ligand-based drug design methods with empirical scoring. Despite advancements in computerized molecular docking methodologies, there remains a gap in optimizing the predictive capabilities of docking simulation software. Accordingly, using the docking scores output by ChooseLD, we evaluated its performance in predicting the bioactivity of G-protein coupled receptor (GPCR) and kinase bioactivity, specifically focusing on Ki and IC₅₀ values. We evaluated the accuracy of our algorithm through a comparative analysis using force-field-based predictions from AutoDock Vina. Our findings suggested that the modified ChooseLD could accurately predict the bioactivity, especially in scenarios with a substantial number of known ligands. These findings highlight the importance of selecting algorithms based on the characteristics of the prediction targets. Furthermore, addressing partial model fitting with database knowledge was demonstrated to be effective in overcoming this challenge. Overall, these findings contribute to the refinement and optimization of methodologies in computer-aided drug design, ultimately advancing the efficiency and reliability of in-silico screening processes.

Single-molecule imaging provides information on diffusion dynamics, oligomerization, and protein-protein interactions in living cells. To simultaneously monitor different types of proteins at the single-molecule level, orthogonal fluorescent labeling methods with different photostable dyes are required. G-protein-coupled receptors (GPCRs), a major class of drug targets, are prototypical membrane receptors that have been studied using single-molecule imaging techniques. Here we developed a method for labeling cell-surface GPCRs inspired by the HiBiT system, which utilizes the high affinity complementation between LgBiT and HiBiT fragments of the NanoLuc luciferase. We synthesized four fluorescence-labeled HiBiT peptides (F-FiBiTs) with a different color dye (Setau-488, TMR, SaraFluor 650 and SaraFluor 720). We constructed a multicolor total internal reflection fluorescence microscopy system that allows us to track four color dyes simultaneously. As a proof-of-concept experiment, we labeled an N-terminally LgBiT-fused GPCR (Lg-GPCR) with a mixture of the four F-FiBiTs and successfully tracked each dye within a cell at the single-molecule level. The F-FiBiT-labeled Lg-GPCRs showed agonist-dependent changes in the diffusion dynamics and accumulation into the clathrin-coated pits as observed with a conventional method using a C-terminally HaloTag-fused GPCR. Taking advantage of luciferase complementation by the F-FiBiT and Lg-GPCRs, the F-FiBiT was also applicable to bioluminescence plate-reader-based assays. By combining existing labeling methods such as HaloTag, SNAP-tag, and fluorescent proteins, the F-FiBiT method will be useful for multicolor single-molecule imaging and will enhance our understanding of GPCR signaling at the single-molecule level.

Biolayer interferometry (BLI) is an optical sensor-based analytical method primarily used for analyzing interactions between biomolecules. In this study, we explored the application of BLI to observe the cleavage reaction of glutathione S-transferase (GST)-tagged fusion protein by human rhinovirus (HRV) 3C protease on a BLI sensor as a new application of the BLI method. The soluble domain of the Tic22 protein from Plasmodium falciparum was expressed and purified as a GST-tagged fusion protein, GST-Tic22, in Escherichia coli. A cleavage sequence for HRV 3C protease was inserted between the GST tag and the soluble domain of Tic22. First, we confirmed that GST-Tic22 was specifically cleaved at the inserted sequence by HRV 3C protease using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following this, GST-Tic22 was immobilized on a BLI sensor, and enzymatic cleavage by the HRV 3C protease was monitored. We observed that the soluble domain of Tic22 was cleaved and released into the buffer over time, and this reaction was dependent on the enzyme concentration. This result demonstrates that the BLI method can be used to evaluate the cleavage of the GST tag by the HRV 3C protease in real time under different conditions. This method enables a more efficient search for the optimal conditions for the tag cleavage reaction in fusion proteins, a process that has historically required a substantial amount of time and effort.

Visceral organs in vertebrates are arranged with left-right asymmetry; for example, the heart is located on the left side of the body. Cilia at the node of mouse early embryos play an essential role in determining this left-right asymmetry. Using information from the anteroposterior axis, motile cilia at the central region of the node generate leftward nodal flow. Immotile cilia at the periphery of the node mechanically sense the direction of leftward nodal flow in a manner dependent on the polarized localization of Pkd2, which is localized on the dorsal side of cilia. Therefore, only left-side cilia are activated by leftward nodal flow. This activation results in frequent calcium transients in the cilia via the Pkd2 channel, which leads to the degradation of Dand5 mRNA only at the left-side crown-cells. This process is the mechanism of initial determination of the left-side-specific signal. In this review, we provide an overview of initial left-right symmetry breaking that occurs at the node, focusing mainly on a recent biophysical study that revealed the function of nodal immotile cilia using advanced microscopic techniques, such as optical tweezers and super-resolution microscopy.

Chemical tongues are emerging powerful bioanalytical tools that mimic the mechanism of the human taste system to recognize the comprehensive characteristics of complex biological samples. By using an array of chromogenic or fluorogenic probes that interact non-specifically with various components in the samples, this tool generates unique colorimetric or fluorescence patterns that reflect the biological composition of a sample. These patterns are then analyzed using multivariate analysis or machine learning to distinguish and classify the samples. This review focuses on our efforts to provide an overview of the fundamental principles of chemical tongues, probe design, and their applications as versatile tools for analyzing proteins, cells, and bacteria in biological samples. Compared to conventional methods that rely on specific targeting (e.g., antibodies or enzymes) or comprehensive omics analyses, chemical tongues offer advantages in terms of cost and the ability to analyze samples without the need for specific biomarkers. The complementary use of chemical tongues and conventional methods is expected to enable a more detailed understanding of biological samples and lead to the elucidation of new biological phenomena.

Plant myosins have higher velocities than animal myosins. Among them, myosins in freshwater algae of the genus Chara have extremely high velocities. We have biochemically studied myosins that perform high-speed movements in the alga Chara. Our studies have elucidated the structural and enzymatic basis for the fast movement of Chara myosins. This review outlines the history leading to the discovery of the fastest myosin, algae Chara myosin XI, and the structure-function correlation of the fastest myosin. This review article is an extended version of the Japanese article, "Structure-function Relationship of the Fastest Myosin" by Ito et al., published in SEIBUTSU BUTSURI Vol. 63, p. 91-96 (2023).

Mycoplasma mobile is a parasitic bacterium that forms gliding machinery on the cell pole and glides on a solid surface in the direction of the cell pole. The gliding machinery consists of both internal and surface structures. The internal structure is divided into a bell at the front and chain structure extending from the bell. In this study, the internal structures prepared under several conditions were analyzed using negative-staining electron microscopy and electron tomography. The chains were constructed by linked motors containing two complexes similar to ATP synthase. A cylindrical spacer with a maximum diameter of 6 nm and a height of 13 nm, and anonymous linkers with a diameter of 0.9-8.3 nm and length of 14.7±6.9 nm were found between motors. The bell is bowl-shaped and features a honeycomb surface with a periodicity of 8.4 nm. The chains of the motor are connected to the rim of the bell through a wedge-shaped structure. These structures may play roles in the assembly and cooperation of gliding machinery units.

Single-molecule imaging in living cells is an effective tool for elucidating the mechanisms of cellular phenomena at the molecular level. However, the analysis was not designed for throughput and requires high expertise, preventing it from reaching large scale, which is necessary when searching for rare cells that induce singularity phenomena. To overcome this limitation, we have automated the imaging procedures by combining our own focusing device, artificial intelligence, and robotics. The apparatus, called automated in-cell single-molecule imaging system (AiSIS), achieves a throughput that is a hundred-fold higher than conventional manual imaging operations, enabling the analysis of molecular events by individual cells across a large population. Here, using AiSIS, we demonstrate the single-molecule imaging of molecular behaviors and reactions related to tau protein aggregation, which is considered a singularity phenomenon in neurological disorders. Changes in the dynamics and kinetics of molecular events were observed inside and on the basal membrane of cells after the induction of aggregation. Additionally, to detect rare cells based on the molecular behavior, we developed a method to identify the state of individual cells defined by the quantitative distribution of molecular mobility and clustering. Using this method, cellular variations in receptor behavior were shown to decrease following ligand stimulation. This cell state analysis based on large-scale single-molecule imaging by AiSIS will advance the study of molecular mechanisms causing singularity phenomena.

This paper describes a method for recording X-ray diffraction patterns from a small amount of fibrous protein materials while being oriented by using a micro shear-flow cell. This cell consists of two concentrically arranged glass tubes. The inner tube is stationary, while the outer one rotates at a high speed. The gap between the two tubes is about 100 μm, into which the suspension of fibrous protein materials is injected. By using synchrotron-radiation X-ray microbeams (diameter, 10 μm), clear diffraction images from oriented protein materials can be recorded. The required volume of the sample is only about 10 μl. This method can also be combined with the laser-flash photolysis of caged compounds. Examples of application of this method to the flagella of a green alga Chlamydomonas, and sperm of a tunicate Ciona are presented.

Blood cancer is a condition in which white blood cells grow uncontrollably. Tumor treating fields (TTF) are a modality of cancer treatment that utilizes electric fields to target malignant cells. To optimize the efficacy of TTF, it is necessary to investigate the distribution of electric field through varying electrode configurations and input parameters. This allows for enhancement of electric field intensity in targeted areas while minimizing intensity in sensitive areas. Analysis of electric field distribution was conducted through simulation of brachial models with varying electrode configurations and input parameters, utilizing the COMSOL Multiphysics 5.4 software. Additionally, investigations were carried out to assess tissue dose density. The dose density value at primary target for all electrode configurations and input parameters do not exceed the threshold value (770 W/m³), whereas the electric field value at the primary target satisfied the threshold value (100 V/m) on the system that used 4 plate-shaped electrodes and arm contour-shaped electrodes with an input voltage of 20 V, and at the input voltage 15 V, only 4 arm contour-shaped electrodes that satisfied the threshold value. An increase in input voltage, electrodes addition, and electrodes adjustment to skin contour shape result in an enhancement of electric field distribution and average electric field value at primary targets.