Biophysica, Volume 5, Issue 1 (March 2025) – 9 articles

ATP analogues are essential tools in enzymology and structural biology, but the structural and functional implications of their chemical modifications on nucleotide-binding proteins are often underappreciated. To address this, we evaluated a panel of ATP analogues, focusing on thiosubstituted and fluorinated molecules, using the AAA+ ATPase p97 as a benchmark system. Hydrolysis stability and impact on protein conformation, binding modes, and kinetics of enzymatic catalysis were assessed by protein-detected methyl NMR and ligand-detected ¹⁹F NMR in solution, as well as ³¹P solid-state NMR of nucleotides within protein sediments. ATPγS and AMP-PNP emerged as the most suitable analogues for preserving pre-hydrolysis states over extended periods, despite undergoing gradual hydrolysis. In contrast, both AMP-PCP and α/β-thiosubstituted analogues failed to induce native protein conformations in p97. Notably, we demonstrate a novel real-time NMR setup to explore the effect of nucleotide mixtures on cooperativity and the regulation of enzymes. Additionally, aromatic fluorine TROSY-based ¹⁹F NMR shows promise for direct ligand detection in solution, even in the context of large macromolecular complexes. These findings provide critical guidance for selecting ATP analogues in functional and structural studies of nucleotide-binding proteins. Full article

Electrostatic charge significantly influences microorganism–surface interactions, including viral adhesion and transmission. While bacterial surface charges are well characterized using electrophoretic mobility and X-ray photoelectron spectroscopy (XPS), similar studies for viruses are limited. This work bridges the gap by estimating the negative surface charge of the Phi6 bacteriophage using XPS data. A novel approach is applied, combining chemical functionalities derived from XPS with a system of equations to quantify surface polysaccharides, proteins, hydrocarbons, and negatively charged groups (RCOO⁻ and R₂PO₄⁻). The results indicate a predominance of proteins on the viral surface and a pH-dependent negative charge: phosphate groups dominate at low pH (1–3), while both groups contribute equally at pH 4–9. These findings provide a deeper understanding of virus–surface interactions and underscore the importance of pH in modulating viral surface charge. This method, which surpasses traditional electrophoretic mobility techniques, offers new perspectives for studying viral adhesion and developing improved antiviral materials and disinfection strategies. Full article

Silver(I) ions and organometallic complexes thereof are well-established antimicrobial agents. They have been employed in medical applications for centuries. It is also known that some bacteria can resist silver(I) treatments through an efflux mechanism. However, the exact mechanism of action remains unclear. All-atom force-field simulations can provide valuable structural and thermodynamic insights into the molecular processes of the underlying mechanism. Lennard-Jones parameters of silver(I) have been available for quite some time; their applicability to properly describing the binding properties (affinity, binding distance) between silver(I) and peptide-based binding motifs is, however, still an open question. Here, we demonstrate that the standard 12-6 Lennard-Jones parameters (previously developed to describe the hydration free energy with the TIP3P water model) significantly underestimate the interaction strength between silver(I) and both methionine and histidine. These are two key amino-acid residues in silver(I)-binding motifs of proteins involved in the efflux process. Using free-energy calculations, we calibrated non-bonded fix (NBFIX) parameters for the CHARMM36m force field to reproduce the experimental binding constant between amino acid sidechain fragments and silver(I) ions. We then successfully validated the new parameters on a set of small silver-binding peptides with experimentally known binding constants. In addition, we monitored how silver(I) ions increased the α-helical content of the LP1 oligopeptide, in agreement with previously reported Circular Dichroism (CD) experiments. Future improvements are outlined. The implementation of these new parameters is straightforward in all simulation packages that can use the CHARMM36m force field. It sets the stage for the modeling community to study more complex silver(I)-binding processes such as the interaction with silver(I)-binding-transporter proteins. Full article

Purpose: The purpose of this study is to analyze the relationship between nuclear charge (Z), atomic mass (A), LET (linear energy transfer for maximal relative biological effectiveness (RBE)) for accelerated ions based on the hypothesis that for each ion, LET_U is related to their nuclear radius. Methods: Published LET_U data for proton, helium, carbon, neon, silicon, argon, and iron ions and their Z and A numbers are fitted by a power law function (PLF) and compared with PLF based on atomic cross-sections and nuclear dimensions for spherical or spheroidal atomic nuclei. The PLF allows for isoeffective RBE estimations for different ions at any value of LET based on the LET_U estimations. For any two ions, A and B, and a specified bioeffect obtained at LET_A, the equivalent isoeffective LET_B, is estimated using ${LET}_B={LET}_A.\left({\displaystyle \frac{{LET}_{U\left[B\right]}}{{LET}_{U\left[A\right]}}}\right)$. Results: The data-fitting program provided the following results: ${LET}_U=78.1.A^{0.26}$, and ${LET}_U=86.6.Z^{0.29}$, where 78.1 and 86.6 keV.μm⁻¹ are the proton LET_U values (i.e., without proton cellular range limit considerations). Goodness-of-fit tests are similar for each model, but the proton estimations differ. These exponents are lower than 0.66 and 0.33 (those for nuclear cross-sections and spherical nuclear radii, respectively), but suggest prolate nuclear shapes in most of the ions studied. Worked examples of estimating isoeffective LET values for two different ions are provided. Conclusions: The fitted power law relationships between LET_U and Z or A are broadly equivalent and compatible with prolate nuclear shapes. These models may offer a more rational basis for future ion-beam radiobiology research. Full article

Cardiovascular diseases have become the leading cause of death in developed countries. Among these, some are related to disruptions in the electrical synchronization of cardiac tissue leading to arrhythmias such as atrial flutter, ventricular tachycardia, or ventricular fibrillation. Their origin is diverse and involves several spatial and temporal scales, ranging from nanoscale ion channel dysfunctions to tissue-level fibrosis and ischemia. Mathematical models play a crucial role in elucidating the mechanisms underlying cardiac arrhythmias by simulating the electrical and physiological properties of cardiac tissue across different spatial scales. These models investigate the effects of genetic mutations, pathological conditions, and anti-arrhythmic interventions on heart dynamics. Despite their varying levels of complexity, they have proven to be important in understanding the triggers of arrhythmia, optimizing defibrillation protocols, and exploring the nonlinear dynamics of cardiac electrophysiology. In this work, we present diverse modeling approaches to the electrophysiology of cardiac cells and share examples from our own research where these approaches have significantly contributed to understanding cardiac arrhythmias. Although computational modeling of the electrical properties of cardiac tissue faces challenges in integrating data across multiple spatial and temporal scales, it remains an indispensable tool for advancing knowledge in cardiac biophysics and improving therapeutic strategies. Full article

Since 2020, the attention of the scientific community has been focused on the overwhelming COVID-19 pandemic, the infectious disease caused by the coronavirus that has affected populations worldwide. The alarming number of deaths and the severity of the symptoms have driven studies aimed at combating this disease. One of the key components in the development of this disease is the protein M^Pro, responsible for the replication and transcription of the virus, making it an excellent biological target in research efforts seeking an effective treatment for the disease. Furthermore, studies have shown that vanadium complexes, such as bis(N′,N′-dimethylbiguanide)oxovanadium (IV), VO(metf)₂∙H₂O, exhibit highly promising effects for the treatment of COVID-19. This molecule contains a ligand known as metformin, which also holds a prominent place as a potential agent in the treatment of this disease due to its antiviral properties. Therefore, an investigation into the interactions between these two systems (M^Pro+Vanadium Complex and M^Pro+Metformin) is pertinent given the significance of these two molecules. Thus, computational studies such as molecular docking and classical molecular dynamics are considered advantageous, assisting in this comparative study, as well as providing a deeper understanding of the interactions that occur within each of them. Full article

Nature equipped red blood cells (RBCs) with diverse mechanical properties, which makes it possible to examine blood with different RBC properties (size, shape, aggregability, deformability). We investigated whether the shelf life of cow blood (stiff RBCs, low aggregability) is longer compared with pig blood (deformability/aggregability comparable to human) due to a delay in RBC clustering and decomposition. Blood was drawn from conscious pigs and cows in their familiar environment to reduce stress and stored 30 days at +7 °C. RBCs remained intact in cow samples whereas pig samples became hemolytic after day 20. White blood cells and platelets decreased with similar percentages in both species. Hematocrit (HCT) decreased due to RBC shrinking in bovine samples and due to RBC decay in porcine samples. Blood viscosity increased in both species although HCT decreased. In porcine samples, shear thinning decreased progressively, indicating a gradual loss of sample cohesion with storage. Yield stress and storage modulus decreased with hemolysis. In HCT-native cow samples, shear thinning, yield stress, and storage modulus showed high intraindividual variability, but the mean values did not change over the time course. In HCT-adjusted (38%) cow samples, solidification occurred after day 7, followed by a reduction in cohesion and shear thinning until the end of storage. Full article

The thermodynamic study of protein folding shows the generation of a narrow range of ΔG° values, as a net result of large changes in the ΔH° and TΔS° values of the folding process. The obvious consequence of this narrow range of values is that a linear enthalpy–entropy relationship, showing apparent enthalpy–entropy compensation (EEC), is clearly observed to be associated with the study of protein folding. Herein, we show the ΔH°, TΔS°, and ΔG° values for a set of 583 data from protein folding processes, at various temperatures, as calculated by using the Gibbs–Helmholtz equations. This set of thermodynamic data was calculated from the melting temperature (Tm), the melting enthalpy (ΔHm), and the change in heat capacity (ΔCp°) values, all of them associated with the heat-induced protein unfolding processes and included in the ProTherm Data Base. The average values of enthalpy (ΔH°av), entropy (TΔS°av), and free energy (ΔG°av) for the folding process were calculated within the range of temperature from 0 °C to the average value of Tm. The values and temperature dependency of TΔS°av within this temperature range are practically equal to those corresponding to ΔH°av, while ΔG°av remains small and displaying a curve with a minimum at about 10 °C and a value of ΔG° = −30.9 kJ/mol at the particular temperature of 25 °C. The large negative value of TΔS°av, together with the also large and negative value of ΔCp°av, suggests large conformational changes and important EEC, thus causing the small average value of ΔG° for protein folding, which is enough to guarantee both protein stability and molecular flexibility to allow for adaptation to the chemical potentials of the environment. Our analysis suggests that EEC may be the quantum-mechanical evolutive mechanism to make functional proteins adaptative to environmental temperature and metabolite concentrations. The analysis of protein folding data, compared with those of protein–ligand interaction, allows us to suggest strategies to overcome EEC in the design of new drugs. Full article

Light microscopy has emerged as one of the fundamental methods to analyze biological systems; novel techniques of 3D microscopy and super-resolution microscopy (SRM) with an optical resolution down to the sub-nanometer range have recently been realized. However, most of these achievements have been made with fixed specimens, i.e., direct information about the dynamics of the biosystem studied was not possible. This stimulated the development of live cell microscopy imaging approaches, including Low Illumination Fluorescence Microscopy, Light Sheet (Fluorescence) Microscopy (LSFM), or Structured Illumination Microscopy (SIM). Here, we discuss perspectives, methods, and relevant light doses of advanced fluorescence microscopy imaging to keep the cells alive at low levels of phototoxicity. Full article