European Biophysics Journal最新文献

Isothermal titration calorimetry (ITC) is the gold standard for thermodynamic characterization of molecular interactions, and more recently has allowed for the provision of kinetic information in a single in-solution and label-free measurement. While extensively applied to protein-protein and protein-ligand interactions, ITC remains underutilized in nucleic acid research, despite its ability to resolve complex molecular binding events. In this study, we leverage ITC to characterize the formation of DNA triplexes, a class of non-canonical DNA structures involved in gene regulation and genomic integrity. Crucially, this study represents the first quantitative determination of kinetic parameters for triplex DNA formation using ITC, offering unprecedented insights into the dynamic aspects of these interactions. By integrating ITC data with global fitting models, we extract both thermodynamic and kinetic parameters, revealing how sequence composition, strand length, and pH influence triplex stability. The exceptional sensitivity of ITC allows us to detect subtle energetic differences that conventional spectroscopic techniques often overlook, establishing ITC as a superior tool for nucleic acid research. Our findings not only validate ITC as an indispensable method for studying triplex DNA but also open new avenues for investigating nucleic acid assembly kinetics with high precision.

The stability of membrane proteins depends on amino acid-specific interactions within the protein structure, its surroundings, e.g. the lipid membrane and ionic solutions, and their substrates. A molecular understanding of the factors affecting protein stability is essential for comprehending the respective structure–function relationships. This study aims to investigate the influence of native cysteine residues, ions, and glycerol on the stability of GlpF, the aqua(glycerol)porin of E. coli, under well-controlled conditions. To this end, the wild-type protein and its variant with four cysteine residues in the transmembrane helix bundle mutated to glycines were overexpressed and purified. The thermal unfolding of GlpF occurs at a transition temperature of 59.2 ± 1.4 °C, corresponding to an apparent Gibbs free energy of unfolding of approximately 10 kcal/mol. In contrast to the wild-type protein, the mutated GlpF exhibits a second unfolding transition, lowered by approximately 10 °C, and a decreased Gibbs free energy of unfolding of 5 kcal/mol. Our findings highlight that divalent ions and glycerol have a more pronounced stabilizing effect on the mutated GlpF than the wild-type protein. The structural consequences of cysteine to glycine mutations in terms of transmembrane helix rearrangement, the inter- and intra-chain H-bond network, and the reduced inter-chain interaction free energy, as well as intra-chain electrostatic energy and van der Waals energy, are revealed by molecular dynamics simulations. In conclusion, our findings illustrate the significance of cysteine residues distant from each other on protein oligomerization and stability, and the amplified impact of (de)stabilizing agents on less stable protein variants.

The overall complexity of organisms increases during the course of evolution, starting with the first self-replicating molecules, followed by prokaryotes and eukaryotes, first unicellular and later multicellular. We present an opinion that non-cellular genetic entities such as transposable elements, plasmids, viruses and viroids, although originally parasitic, selfish and sometimes destructive elements, may contribute to the increase of complexity. We propose that non-cellular genetic elements impose (parasitic) pressure on the cooperative genes of cellular organisms, driving the sequence of evolutionary transitions from the first cooperative replicators to multicellular life forms, and have suggested that they belong to a separate kingdom of life, the Acytota. The complexity increase is probably caused by the high proliferation capacity of these non-cellular genetic elements, their frequent horizontal gene transfer, participation in parasite-host arms races, formation of epigenetic silencing mechanisms as well as the ability to build genetic regulatory networks. Simultaneously, these elements contribute to complexity by supplying genetic material via domestication, genome rearrangements, and dispersal of regulatory elements. Complexity has not only increased during evolution, there are also examples of simplification, both during chemical evolution (in prebiotic chemistry) and the evolution of parasites. Therefore, we describe the ups and downs of organism complexity and discuss the reasons for the general dominant upward trend, namely coevolution and the interaction of existing modules.

The ordered mesoporous silica SBA-15 is a promising platform for protein immobilization and delivery, since adsorption into its porous matrix may improve protein structural stability. Here, three proteins—myoglobin (Mb), concanavalin A (ConA), and soybean trypsin inhibitor (STI)—were loaded into conventional SBA-15 particles with an average mesopore diameter of 9.3 nm to investigate structural changes in their predominant content after adsorption. Powdered biocomposites were prepared by loading each protein at two silica-to-protein weight ratios (10:1 and 5:1). Protein structures were evaluated by synchrotron radiation circular dichroism and fluorescence spectroscopy following resuspension in buffer solutions (pH 7.0 and 2.2). The structural changes were qualitatively analyzed and found to be more influenced by the silica-to-protein ratio for Mb and ConA, while buffer pH had a lesser effect on the proteins. A 10:1 ratio better preserved the α-helical spectral features of Mb, whereas a 5:1 ratio favored the retention of the β-sheet signature of ConA; in both cases, the aromatic residue microenvironment remained largely preserved. In contrast, STI, rich in irregular structures, exhibited smaller spectral changes relative to its native structure in both ratios, while the aromatic residue was less exposed to the solvent. Overall, the results reinforce the potential of SBA-15 as a protein carrier by retaining the main features of the protein's secondary structure under specific conditions. The simultaneous comparison of three structurally distinct proteins reveals variable adsorption behaviors and highlights the silica-to-protein ratio as a protein-dependent parameter, reinforcing the importance of optimizing adsorption conditions to develop more stable silica–protein systems.

The Hfq protein is not only a mediator of RNA metabolism but also a key structural element involved in nucleic acid shaping. Its ability to compact and organize DNA, as well as its influence on the dynamics of various DNA-related processes, makes Hfq a central player in the regulation of bacterial chromosomal architecture and function. We previously demonstrated that different DNA methylation states affect Hfq binding and mobility. In this study, we show that Hfq, through its C-terminal region, can influence a DNA entangled/disentangled transition and examine the impact of DNA methylation on this previously uncharacterized function of Hfq. This discovery provides new insights into the role of Hfq in DNA transactions, with potential implications for essential cellular processes such as recombination and replication. Furthermore, this study demonstrates that Synchrotron Radiation Linear Dichroism (SRLD) is a powerful tool that can follow cooperative vs non-cooperative protein induced DNA structural transitions.

Multicellular structures, including cell sheets, are actively used as model systems to study intercellular interactions and can be applied in different areas of regenerative medicine. In this paper, we present a novel approach for measuring mechanical properties of cell sheets based on a simple experimental setup and numerical simulations. The advantage of the present approach is the relative ease of the sample preparation, while previous systems for the tensile tests required specialized and sensitive equipment. With the developed approach, the cell sheet on a polymer membrane is mounted in the holder on one side, and then deflection of the free end of the membrane is measured. The deflection of this cantilever-like construction depends on the elastic modulus of the membrane (which is known) and cell sheet, and also from the traction force generated by the cell sheet. By involving an experimental step with relaxing the traction force and by conducting finite element simulations, both traction force and elastic properties of the cell sheet can be estimated. We performed such measurements on cell sheets from keratocytes from corneal explants and confirmed that the developed approach is applicable for measurement of both traction force and elastic modulus of cell sheets.

Biophysics is an interdisciplinary science that applies the theories and methods of physics to understand biological systems. It encompasses a wide range of topics, from the molecular mechanisms within cells to the physical properties of organisms and ecosystems. The goal of biophysics is to uncover the physical principles underlying the structure and function of biological molecules, cells, and cellular systems, providing a deeper understanding of life itself. The Institute of Biophysics, Czech Academy of Sciences (IBP) stands as a beacon of excellence in the field of biophysical research in the Czech Republic. This article delves into its history, structure, research areas, and major scientific achievements, highlighting the role of IBP in the global scientific community.

The 26th International Analytical Ultracentrifugation Workshop and Symposium (AUC2024) took place at the scenic Banz Abbey near Bad Staffelstein, Germany, from July 22 to 27, 2024. 84 participants from 16 countries (Belgium 1, Canada 7, China 3, Colombia 1, Czech Republic 3, Finland 1, France 3, Germany 35, Israel 1, Italy 1, Japan 2, New Zealand 2, Spain 1, Switzerland 4, United Kingdom 5, United States 14) travelled to Germany to present and discuss the latest advances in the field. 40 workshop sessions were held by world-leading experts covering all aspects of AUC including experimental design, data analysis and data processing according to good manufacturing practice (GMP), but also complementary methods such as hydrodynamic modelling, isothermal titration calorimetry and small angle X-ray scattering data processing were considered. A visit to a stalactite cave in Franconian Switzerland and the Bavarian beer museum in Kulmbach offered a welcome change to the scientific program and started off the 3-day symposium. The presentations featured of course AUC, but also dynamic and static light scattering, small angle X-ray and neutron scattering, surface plasmon resonance, field flow fractionation, calorimetry, chromatography, and electron microscopy. The AUC2024 special volume provides a comprehensive overview of the sustained innovation, utility and relevance of AUC and related solution biophysical and particle technology methods across various disciplines, including biochemistry, structural biology, synthetic polymer chemistry, carbohydrate chemistry, protein and nucleic acid characterization, nano and colloids science, and macromolecular interactions.