Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology最新文献

Rhodopsins are photosensitive transmembrane proteins found in bacteria, archaea, and eukaryotes. Upon photon absorption, they undergo conformational changes critical for their physiological functions, such as ion transport and signal transduction. Microbial rhodopsins are key tools in optogenetics, with applications in biomedical research and clinical treatments. To advance optogenetic techniques, it is crucial to understand the molecular mechanisms of the rhodopsin photocycle. The key approach for studying these mechanisms, cryotrapping, has a setback associated with possible deterioration in resolution due to the unexpectedly large light-induced structural changes. To estimate the scale of these changes, small-angle X-ray scattering (SAXS) can be used. In this study, we applied SAXS to the investigation of light-induced structural changes in purple membranes (PMs) as their two-dimensional crystal organization makes them a suitable model object for studying light-induced changes in 3D rhodopsin crystals. SAXS data from illuminated and non-illuminated PM samples revealed detectable changes, including variations in membrane thickness and planar unit cell dimensions. Particularly, we observed a statistically significant increase in the radius of gyration for flat particles (Rₜ) of ~0.2 Å for the PMs with mutant HsBR_E204Q, while no significant change was detected for wild-type HsBR. However, wild-type HsBR data showed a statistically significant shift in the (1, 1) Bragg peak position (Δ q ~ –10^–4 Å^–1; ~3% of the peak width) upon illumination. For HsBR_E204Q data, the shift was two times greater; however, in this case due to the lower sample concentration ε_Δq was ~50%, making it difficult to compare the wild-type and mutant cases. Future experiments should aim for better signal-to-noise ratios using synchrotron radiation, higher sample concentrations, and longer exposure times.

Unnatural amino acids (UAAs) are powerful tools for investigating protein structure and dynamics. Their inclusion in a protein can help reveal important details about its functions and structural features, especially with respect to membrane proteins. However, efficient incorporation of UAAs into membrane proteins remains a challenge. In this review, we explore using Leishmania tarentolae (LEXSY) as a host system to express membrane proteins with incorporated UAAs, aiming to develop a versatile platform for future research and drug discovery.

IRR is a member of the insulin receptor (IR) family that has no known endogenous peptide agonists but can be activated by weak alkaline environments and has thus been proposed to function as an extracellular pH sensor. IRR activation by alkali is determined by its N-terminal extracellular region. Using recently published Cryo-EM structure of IRR ectodomain we identify three amino acids (R87, K143 and S516) that may be involved in the IRR activation by alkali. In this report we show that the triple substitution of these amino acids with alanine leads to complete disappearance of the IRR pH sensitivity. Thus, these amino acids can form one of the possible pH sensor sites in the IRR molecule.

Receptor for advanced glycation endproducts (RAGE) plays an important role in the development of inflammation and neurodegenerative diseases. There is no expression of RAGE in healthy cells, but it increases during inflammation processes that leads to tissue damage. RAGE has many ligands of different classes but with similar properties, so RAGE acts as a pattern-recognition receptor. The structure of RAGE lacks information about the transmembrane domain which is necessary for signal transduction by the receptor. In this work, for the first time, using the cell-free expression method, we obtained and purified isotope-labeled fragment of RAGE (residues 335–368, corresponding to the RAGE transmembrane domain flanked by short juxtamembrane regions). For investigation of oligomerization processes we introduced a point oncogenic mutation G349R that is located in the conservative oligomerization motif GxxxG. Nuclear magnetic resonance (NMR) studies of both peptides incorporated into dodecylphosphocholine (DPC) micelles indirectly reveal changes in protein structure and oligomerization properties following the introduction of the oncogenic G349R mutation, which is thought to affect RAGE signaling.

Here we explore capabilities of size exclusion chromatography small-angle X-ray scattering method in case of horse spleen apoferritin solution as an example of a system with high oligomerization heterogeneity. For interpretation of the experimental data, we considered models of polydisperse solution containing monomers, dimers, and trimers of apoferritin globules. Dimers were defined only by intercenter distance L, while trimers were characterized by both L and apical corner value φ. We showed that a reasonable description of the major part of the chromatogram could be reached using the model containing monomers, dimers and one type of trimers with φ about 110°–120°. In the case of two types of trimers in the model, optimal φ values were 110°–140° and 90°–110°; however, the approximation results were considered as ambiguous. Validation of the preferred model (L = 12.5 nm, φ = 120°) showed insufficient description of the experimental data for the left part of the chromatogram (elution volume <12.2 mL) indicating the presence of aggregates in these fractions. Application of the method provided the opportunity to calculate concentrations for oligomers consisting of less than four apoferritin globules, and select averaging range for different fractions properly.

Nowadays, only a few structures of rhodopsins bound with carotenoid were obtained and functionally characterized. Nevertheless, rhodopsins-carotenoids complexes are promising molecular systems for understanding the light-harvesting process in bacteria since such complexes show ability to absorb a significant part of solar energy coming to the Earth surface. The understanding of energy absorption and transfer mechanisms in the carotenoid-retinal complex of rhodopsin would provide significant progress in different areas of modern bioscience such as ecology, protein engineering, optogenetics, etc. Here we present a long (1 µs) molecular dynamical investigation of the structure of microbial rhodopsin from an Antarctic bacterium Hymenobacter psoromatis (strain PAMC26554) (HbR1) and zeaxanthin. The structure of rhodopsin was predicted with a new multi-modal foundation for molecular structure prediction Chai-1 and used for classical MD validation in a 10 × 10 × 12 nm box in a hydrated explicit lipid bilayer. Our simulations show tight binding of the carotenoid and rhodopsin with the average distance equal to 0.25 nm between the closest atoms of carotenoid and retinal.

In this study, we investigated thermosensitive histidine kinase DesK from Bacillus subtilis with the aim of obtaining a monodisperse protein preparation suitable for further structural studies. A homogeneous sample, both in terms of oligomeric state and ligand binding, is favorable for electron microscopy or X-ray diffraction applications. The binding of ATP is required for autophosphorylation activity of DesK, thus we aimed at developing heterologous expression techniques that would yield ATP-bound protein. We focused on two critical factors that may influence ATP binding: the nutrient composition of the growth medium and the expression temperature. Protein characterization was performed using size-exclusion chromatography, with absorbance monitored at both 280 and 260 nm to assess protein homogeneity and nucleotide binding. Our results indicate that while nutrient composition had a negligible effect on the relative amount of ATP-bound protein, expression temperature played a significant role. Specifically, lowering the expression temperature from 37°C to 20°C at the time of induction markedly increased the proportion of ATP-bound DesK oligomers.

This paper presents a statistical analysis and comparison of all existing as of November 20, 2024 membrane protein structures that are represented in the most widely used databases mpstruc, Orientations of Proteins in Membranes (OPM), SCOP and PDBTM, with the total number of structures now reaching 13956. We evaluated the resolution dependencies of structure-derived membrane proteins on their size-related physical characteristics, such as molecular weight, radius and size of the hydrophilic part. Based on the obtained dependencies, we compared the resolving methods of X-ray diffraction and electron microscopy structures, and also compared the methods of protein crystallization in surfo and in meso. The change over time in the number of membrane protein structures obtained by different methods was also examined to analyze trends in structural biology. In 2024, cryo-electron microscopy has emerged as the dominant method, contributing to nearly half of the solved structures, now representing 49.6% of the total. The obtained results demonstrate that the resolution of structures obtained by X-ray diffraction on protein crystals, on average, tends to worsen with increasing protein size, while in electron microscopy, which has recently gained enormous popularity, there is no such trend, but the average resolution is worse. It was also shown that the character of the dependence of resolution on protein size is the same (within error) for the two most common methods of membrane proteins crystallization: in lipid cubic phases (in meso) and in detergent micelles (in surfo). Thus, statistically, neither of these two methods can be considered a priori preferable for achieving better resolution.

Bacterial biosensors based on the Escherichia coli and Bacillus subtilis whole cells are used to study toxicological characteristics of biologically active compounds and toxicants, and are applicable for ecological studies. This work describes the trials of lux-biosensors in the marine research expedition in the northern seas: Barents, Kara, and Laptev. The presence of toxicants in samples of bottom sediments and tissues of marine invertebrates was estimated by the activation of stress promoters in biosensor cells. It was shown that the collected samples generally did not show toxicological characteristics in terms of the ability to activate oxidative stress and SOS response. However, several samples were able to activate transcription of the DNA glycosylase promoter in biosensor bacterial cells. This effect was shown for some samples of bottom sediments and arthropod tissues from Mesidothea sabini var. sabini., Pycnogonida and some Amphipoda.