Biophysical reviews最新文献

This review aims to provide evidence that the fibrinogen-like structures of invertebrates (on the example of the intracellular coagulation protein coagulogen from horseshoe crabs) and fibrinogen of vertebrates (such as lampreys, which are among the most primitive extant vertebrates, as well as higher-ordered animals on the phylogenetic evolutionary scale, such as chickens and humans) reflect their methionine-dependent adaptation to the steady-state level of reactive oxygen species (ROS). Since methionine residues disposed in primary structure of proteins may serve as ROS interceptors, the absence of methionines in the coagulogen structure suggests that it is not subject to ROS attack in the same extent as vertebrate extracellular fibrinogen. The structures of vertebrate fibrinogens (such as the central E-nodule composed of the NH₂-terminal portions of all six chains of the fibrinogen molecule, β-, and γ-nodules consisting of the C-terminal parts of the β and γ chains, respectively, and α-helical coiled-coil connectors, which hold the central and distal nodules of fibrinogen together) have minor differences among distant species. With the exception of the E-nodule, each of these conserved, homologous structures of vertebrate fibrinogens exhibits a similar distribution of methionines, including surface-exposed ones. It is suggested that the structures of the variable αC regions evolved to enhance their role in not only the fibrin assembly process but also the antioxidant protection of the central E-nodule. This implies that evolutionary processes of fibrinogen structure in different species and methionine-dependent defense are intrinsically linked.

Classical force fields remain widely used in molecular modeling due to their efficiency but fail to accurately capture reactivity and complex environments. Quantum mechanical methods like DFT offer higher accuracy but are computationally prohibitive for large biomolecules. Machine learning interatomic potentials (MLIPs) bridge this gap by approximating potential energy surfaces with near-DFT precision while enabling large-scale simulations. MLIPs learn directly from quantum data and can generalize across diverse chemical environments. This review outlines the theoretical basis of MLIPs, including training on energy and force data, symmetry constraints, and common architectures-ranging from descriptor-based to graph-based and equivariant neural networks. Key applications are examined in biomolecular contexts: conformational sampling, enzymatic catalysis, and ligand binding. Integration with molecular dynamics packages like OpenMM and LAMMPS is increasingly streamlined. While challenges remain-such as generalization to out-of-distribution systems, limited interpretability, and data scarcity-ongoing advances in datasets, hybrid modeling, and infrastructure are rapidly improving practical adoption. MLIPs represent a major step forward in atomistic simulations and are poised to become central tools in structural biology, enzymology, and computational drug discovery.

Cellular activity depends on constant flux of ions across biological membranes. Artificial membrane models like planar lipid bilayers and liposomes are ideal for studying membrane transport phenomena as they are free of the structural complexity of cells and allow examination of transport processes under tightly controlled conditions. Over the last decades, artificial membrane-based techniques like single-channel recording and fluorescent monitoring of transport through bulk lipid vesicle suspensions have revealed many molecular mechanisms of transport. Recently, giant unilamellar vesicles (GUVs), cell-sized liposomes, have emerged as an important tool for studying cellular processes, including ion transport. The principal advantage of GUVs derives from their micron scale, which enables ease of visualisation and manipulation using microscopy and microhandling. For that reason, GUVs have also become the state-of-the-art for recapitulating a host of cell structures and functions for the purpose of developing artificial cells. Taken together, GUVs represent a promising biomimetic system to elucidate ion transport mechanisms and unravel the association between ion fluxes and various cellular processes such as neuronal transduction, nutrient uptake, electrochemical gradient development. Nevertheless, despite their great potential as a model system, the use of GUVs in ion transport studies is still limited. The aim of this review is to outline recent GUV-based ion transport studies, describe the current techniques for measuring ion transport in GUVs, compare the utility of GUVs relative to other available techniques such as single-channel current recording, and explore the potential of using GUVs to investigate complex ion transport processes.

Despite more than a century of research into the neuromuscular junction, it still remains a widely used experimental model for studying molecular mechanisms of synaptic transmission in various physiological and pathological conditions, as well as for testing different pharmacological agents. One of the generally accepted methodologies for achieving these goals is electrophysiological recordings using microelectrode techniques. Application of extracellular microelectrodes makes it possible to precisely evaluate the parameters of presynaptic action potential and its coupling with evoked exocytotic event. The use of intracellular microelectrodes allows the measurements of a resting membrane potential, amplitude-temporal parameters of evoked and spontaneous postsynaptic potentials, which depend on both neurotransmitter release and the postsynaptic membrane sensitivity to neurotransmitter. A voltage-clamp technique with two intracellular microelectrodes is the best option to access postsynaptic currents for characterization of both quantal content and functioning receptor-channel complexes within the end-plate region. To apply all of the above microelectrode approaches under conditions of motor nerve stimulation, an immobilization of neuromuscular preparations is necessary. Skeletal muscle paralysis can be accomplished by decreasing neurotransmitter release (via lowering extracellular Ca²⁺ and elevating external Mg²⁺ levels), a partial blockage of nicotinic acetylcholine receptors, a selective inhibition of skeletal muscle-specific voltage-gated sodium (Na_v1.4) channels, a reduction of membrane potential below the excitation threshold (due to a transverse dissection of the muscle fibers), uncoupling excitation-contraction (by hyperosmotic shock), and inhibiting skeletal muscle myosin ATPase. All of the listed methods have their advantages and disadvantages, which are discussed in this review.

Failure of functions of CFTR (cystic fibrosis transmembrane conduction regulator) gene, which encodes a protein of a selective ion channel, is causing cystic fibrosis. Cystic fibrosis is a severe systemic monogenic disease with an autosomal recessive type of inheritance, which significantly reduces the duration and quality of life of patients. It is one of the most common hereditary diseases. Studying of molecular functions of CFTR protein in different types of cells, its structural and functional network interactions are critically important for the development of a new and more effective pathogenetic therapy. We are reviewing papers on the structure of the CFTR protein and its pathogenic genetic variants, as well as methods of pathogenetic therapy of cystic fibrosis by CFTR modulators and gene engineering. Recent gene engineering approaches to keep CFTR functions are discussed, such as gene-replacement therapy and genome editing, as well as viral and non-viral delivery systems and strategies of genomic editors.

To change shape, move, grow and divide, cells employ various motor and non-motor proteins that convert chemical energy into the generation of mechanical force. Force spectroscopy tools that allow the measurement of these forces generated by individual molecules revolutionised our understanding of single-molecule mechanics over the past three decades. These techniques, however, remain largely confined to studies with purified components outside cells. A critical, unresolved challenge lies in deciphering how these force-generating and force-sensing molecules coordinate their activities inside living cells. In this review, we discuss advances in magnetic tweezers designed to measure and apply mechanical forces intracellularly. We highlight recent progress in magnetic tweezers that began to provide an understanding of how active mechanical forces drive rearrangements of biological structures. We also discuss challenges associated with applying forces locally and precisely. We identify two key areas that hold potential for the development of tools for direct mechanical manipulations of specific molecules inside living cells: (1) instrument design to generate and control magnetic gradients at the single-cell scale, and (2) development of magnetic biofunctionalised particles capable of targeting specific structures. The integration of these advances should enable unprecedented ability to manipulate intracellular forces, opening new avenues to study intracellular organisation, mechanotransduction pathways, cell division and migration. By addressing current limitations in specificity and resolution, next-generation magnetic tweezers may finally bridge the gap between single-molecule biophysics in vitro and cell-scale mechanobiology in living cells.

Due to its important physiological role and ease of isolation, hemoglobin has become one of the most studied proteins. One of the most famous researchers of hemoglobin in the early twentieth century was Gilbert Smithson Adair (1896-1979), who published a series of six papers devoted to this protein. This review, timed to coincide with the 100th anniversary of the publication of this series, highlights Adair's fundamental contribution to the study of the hemoglobin molecule and the mathematical description of oxygenation. The results of Adair's experiments determined the further course of hemoglobin research, and the equation he proposed became the basis for the construction of structural and functional models of oxygenation later proposed by Pauling (PNAS 21(4):186-191, 1935) and Koshland et al (Biochemistry 5(1):365-385, 1966). This review briefly discusses the background of Adair's experiments and theories, as well as issues related to the mathematical interpretation of this equation, its approximating properties, and the interpretation of its coefficients.

X-ray diffraction enables determination of biomolecular structure but requires well-diffracting crystals that are notoriously difficult to grow. Porous materials can aid the crystallization of refractory proteins and, since knowledge of the mode of action of such materials may contribute to finding new crystallization inducers, this process has been studied thoroughly. It was established that, even under conditions where heterogeneous nucleation on flat surfaces is absent, a synergistic diffusion-adsorption effect inside a sufficiently narrow pore can increase the protein concentration to a level sufficient for crystal nucleation. The formation of a protein crystal in a pore begins with the assembly of molecules into a crystalline layer of monomolecular thickness, which is stabilized by its cohesion with the pore wall. We highlight thermodynamic considerations that provide an estimate of the importance of the protection due to the pore walls for crystal stability. In addition, molecular-kinetic considerations reveal further details of protein crystal nucleation assisted by porous materials. The observation that protein crystals nucleated by means of porous materials often display improved X-ray diffraction is of practical importance for structural studies. It is hoped that this review will guide scientists in their efforts to grow crystals of target proteins, complementing the usual trial-and-error strategies.

Protein synthesis is a fundamental biological process universally mediated by ribosomes-complex ribonucleoprotein assemblies responsible for translating genetic information into functional proteins. Despite significant structural information provided by X-ray crystallography and cryo-electron microscopy (cryo-EM), certain dynamic features of ribosomal function, particularly those involving RNA conformational flexibility and transient interactions, remain challenging to characterize. Electron Paramagnetic Resonance (EPR) spectroscopy, combined with site-directed spin labeling (SDSL), has emerged as a robust complementary approach for probing structural dynamics and conformational heterogeneity in ribosomal complexes. This review summarizes recent advances in applying EPR spectroscopy, particularly pulse dipolar EPR (DEER/PELDOR), to investigate human ribosomal complexes. We discuss methodological aspects of spin-labeling strategies for mRNA, comparing various nitroxide-based labels and highlighting their specific advantages for probing ribosomal interactions. Through representative examples, we illustrate how different EPR techniques yield complementary structural information in studying ribosome-RNA interactions. Key findings include the identification of alternative mRNA conformations within ribosomal complexes, characterization of labile RNA binding sites near the mRNA entry channel, and elucidation of stabilization effects mediated by tRNA interactions. Furthermore, we demonstrate how the integration of EPR data with molecular modeling facilitates accurate interpretation of distance distributions, enabling the correlation of experimental findings with atomic-level structural models. Finally, we address current methodological limitations of EPR spectroscopy, outlining promising perspectives and anticipated advancements in this evolving field.

Purified proteins are sitting in a mostly aqueous environment, with normally some buffer and salt making up the conditions. This is vastly different from their natural habitat, and protein are often affected by this difference, showing signs of destabilisation or aggregation. A common method to improve the protein solubility and homogeneity is adding small molecules to the buffer conditions, as these can aid protein stability and keep the protein in solution at a concentration which is within that needed for the experiments that are to be undertaken. This review is detailing some of the small molecules that are routinely used, with a focus on them being readily available and affordable for all labs. Some of the more common small molecule additives described in this paper are (1) amino acids, like arginine or glycine, (2) sugars, like sucrose, or (3) other osmolytes, such as glycerol. The second part is covering some of the methods that can be utilised to determine whether a small molecule improves the stability of a particular protein.