Biophysics and physicobiology最新文献

Oligomers of amyloid-β (Aβ) peptides are related to Alzheimer's disease, and their formation is accelerated at hydrophilic-hydrophobic interfaces. We performed all-atom molecular dynamics simulations of Aβ(29-42) peptides in bulk water and at an air-water interface. In bulk water, the fragments formed stable aggregates, and the secondary structures were hardly changed. At the interface, the peptides were more easily separated from each other due to the low free-energy barrier and changed their secondary structures more frequently. This conformational flexibility is likely to promote amyloid fibril growth, suggesting a key role of interfacial environments in early aggregation processes.

Neorhodopsin (NeoR), an enzymerhodopsin, has unique molecular properties. NeoR absorbs near-infrared light whose λ_max is located at 690-700 nm. Upon light absorption, NeoR is photoisomerized into the 7-cis form with low quantum yield. Unique color-tuning and photoreaction mechanism of NeoR is owing to the residues surrounding the all-trans retinal chromophore. In NeoR, three carboxylates, E136, D140, and E262, constitute a counterion triad near the retinal Schiff base, and E141 is located near the β-ionone ring. Despite recent experimental and theoretical studies, protonation states of the highly conserved four carboxylates have never been experimentally determined. In this study, we performed comprehensive mutation analysis of NeoR by UV-visible and FTIR spectroscopy. We prepared E136Q, D140N, E141Q, and E262Q mutants of an NeoR. Among four mutants, only E262Q did not form a pigment, suggesting that E262 is the principal Schiff base counterion. Light-induced FTIR spectroscopy detected two bands of protonated carboxylic acids, and vibrational bands were identified as protonated D140 and E141. Hydrogen bond of D140 is strong in NeoR, which is further strengthened upon photoisomerization into the 7-cis form. The Schiff base is the possible hydrogen-bonding partner of D140. Hydrogen bond of E141 is very weak in NeoR, but E141 newly forms a hydrogen bond upon 7-cis isomerization. Protonation states of E136 and E262 were not determined conclusively, whereas the present FTIR study suggests that one negative charge is delocalized at E136 and E262 that contributes to unusual spectral red-shift in NeoR. Four carboxylates near the retinal chromophore in NeoR play their own roles in unique color-tuning and photoreaction mechanism.

Protein loops play crucial roles in the formation of binding and enzyme active sites. However, general structural and biological characteristics of these loops remain unclear. In this study, we investigated loops from structural and evolutionary perspectives using the entire protein data bank (PDB), Homo sapiens, and Escherichia coli proteins. We found that loop sequences tended to be unique among species. However, loop properties exhibited high similarity or conservation. Class, Architecture, Topology, and Homologous superfamily (CATH) classification analysis, which clusters domains within protein chains into superfamilies indicating an evolutionary relationship, suggested that the terminal residues of most loops connected to the same superfamily. The functions of conserved loops were not consistently conserved. The amino acid composition profiles showed different preferences. Collectively, this study provides an overview of loops from structural, functional, and evolutionary perspectives and a vast natural loop repertoire for future investigations.

UV-sensitive cone visual pigments are widespread among vertebrates, including birds, fish, and rodents such as mice, and play essential roles in non-primate vision. Unlike visible-light pigments, which contain a protonated retinal Schiff base and absorb at 400-700 nm, UV pigments maintain a deprotonated Schiff base, but how this state is stabilized has remained unresolved. Here, we applied low-temperature FTIR spectroscopy to the mouse UV-sensitive cone visual pigment (MUV), capturing structural changes associated with Batho intermediate formation. Spectral features characteristic of a deprotonated Schiff base were observed in both the initial and Batho states. Importantly, analysis of the Glu113 mutant demonstrated that Glu113 is protonated under these condition. Combined with the analysis of protein-bound water signals, these results indicate that the hydroxyl group of Glu113 serves as a direct hydrogen-bond donor to the deprotonated Schiff base. Moreover, the Glu113 C=O stretching vibration appeared at an unusually low frequency, revealing the presence of an exceptionally strong hydrogen bond with its surrounding protein environment. Comparison with bovine rhodopsin and cone pigments further revealed that MUV binds fewer water molecules near the retinal. This reduction in hydration suggests that a more hydrophobic environment contributes to lowering the Schiff base pKa and stabilizing its deprotonated state. Sequence analyses across species support this view, highlighting conserved nonpolar residues in transmembrane helix 2 (TM2) that likely disrupt hydrogen-bonding networks and promote UV sensitivity. Together, these findings establish MUV as a model for understanding how specific amino acid environments and hydration patterns enable UV vision in vertebrates.

OLPVR1 is a viral channelrhodopsin found in giant viruses, which can be a useful optogenetic tool for calcium-regulated cell functions. Determination of the open-state structure leads to one of the best understood channelrhodopsins. Recent FTIR spectroscopy of OLPVR1 reported unique structures in the resting and photoisomerized states at 77 K. Here we attempted to obtain difference FTIR spectra of mutants in OLPVR1, where we focused six key residues, S11, E44, E51, D76, D200, and N205. We prepared S11T, E51D, E51Q, N205Q, E44Q, D76N, and D200N, to which spectroscopic analysis was applied. From the λ_max values of D76N and D200N at pH 7 and 8, it was concluded that D200 is the primary counterion. FTIR spectroscopy showed that the carboxylic C=O stretch of WT at 1722 (-)/1707 (+) cm^-1 in H₂O, and at 1718 (-)/1698 (+) cm^-1 in D₂O disappeared in E51Q, from which protonation of E51 was concluded. S11T and N205Q alter the C=O stretch frequency of E51 in the dark, but not in the photoisomerized state at 77 K. This observation implicates that E51 interacts with S11 and N205 through a water-containing hydrogen bond, whereas the interaction is broken by light. Recent crystallographic study visualized flipping motion of E51 upon opening state of OLPVR1, and the present FTIR study reports the removal of E51 from the network with S11 and N205 occurring even at the primary photoreaction stage. Retinal photoisomerization accompanies hydrogen-bonding switch of E51, leading to channel opening of OLPVR1 in late timescale.

The emergence of catalytic RNAs (ribozymes) may have set the stage for an "RNA world" preceding protein evolution. The probability of ribozyme emergence and maintenance would have depended on available oligonucleotide compositions. Excessively high or low sequence diversity could hinder ribozyme formation, whereas balanced diversity is likely more favorable. Multiple steps of chemical evolution-from nucleotide supply and oligomerization to subsequent copying and assembly through nonenzymatic reactions-likely shaped oligonucleotide diversity. In this review, we discuss how oligonucleotide chemical evolution may have involved both selective enrichment and diversification of sequence compositions, with their interplay generating oligonucleotide pools of varying diversity across environments and evolutionary timescales. Current experiments on nonenzymatic RNA-based reactions remain limited to short timescales, but strategies combining DNA and protein enzymes could provide efficient models to investigate the compositional dynamics of oligonucleotides.

The malignancies of prostate tumour cells are assessed by pathologists as grade groups (GGs) from 1 (least aggressive) to 5 (most aggressive) on histopathology images. GGs are associated with the degree of tumour cell differentiation and may have different self-similarities depending on GG and tumour-related cell types, which are neoplastic epithelial, inflammatory, connective tissue, necrotic, and non-neoplastic epithelial cells. We investigated the associations between GGs and fractal dimensions (FDs) for five types of prostate tumour-related cells using a multiple-threshold box counting algorithm (MTBC). We showed the association of FDs of 9 channel images (eosin, hematoxylin, normalised images for red, green, and blue colour channels) with multiple threshold values on histopathology images (patch images) and the feasibility of FD-threshold images in an artificial intelligence model to classify patients into low (GG≤3) and high (GG≥4) GGs. We constructed FD-threshold images based on MTBC algorithm for characterizing prostate tumour cells. A shallow-convolutional neural network (sCNN) model to classify patients into low and high GGs was trained with input data of the FD-threshold images for all 9 channels and evaluated using the area under receiver operating characteristic curve (AUC). There were statistically significant correlations between the FD of non-neoplastic epithelial cells and GG [Pearson correlation coefficient=-0.849, p=0.001]. Significant correlations also existed for connective tissue and the original images. The AUC for the sCNN classification model into high and low GGs was 0.811. FD can characterise physical properties of prostate tumour-related cells for low and high GGs.

Translation-the process by which mRNAs are decoded into nascent peptides-is fundamental to life. This process is regulated by various RNA-binding proteins (RBPs), which interact with their target mRNAs. While traditional biochemical approaches have provided valuable insights into translational control, they rely on ensemble measurements of bulk mRNAs in test tubes. Consequently, the behavior of individual mRNAs and their spatiotemporal dynamics in cells during translational control remain largely unexplored. Offering a way to address such limitations, we developed a method for imaging translational control by Argonaute (AGO) proteins, a class of RBPs, at single-mRNA resolution in cells. This method, which employs three-color fluorescence microscopy to detect mRNAs, nascent peptides, and AGO, can also serve as a versatile platform for analyzing translational control by other RBPs. In this protocol, we provide a step-by-step guide for implementing this method to facilitate spatiotemporal studies of translational control at the single-cell and single-mRNA levels.

Single epidermal keratocytes, which are responsible for wound repair in fish, migrate while maintaining their characteristic shape: a frontal crescent-shaped lamellipodium and a posterior rugby-ball-shaped cell body. These cells are widely used in cell migration studies, especially to examine the role of actin polymerization at the leading edge of the cell. In the posterior part of the cell, stress fibers, which are bundles of actomyosin, are aligned along the seam of the 'rugby ball.' The ball rotates with the stress fibers during migration. The linear contraction of stress fibers appears to drive the rotation of the cell body. This review describes a conversion mechanism from linear motion to rotation driven by stress fiber contraction and soft cell body deformation, which is not found in man-made machines. We also describe a unique research method that is able to demonstrate this machinery by creating robot models. Due to their high migration rate and ease of culturing, fish keratocytes appear to be one of the best materials for studying both single cell and collective cell migration. In this review, we will also give some recent research examples of collective migration using keratocytes.