Biophysica

Cryo-electron microscopy single-particle analysis (cryo-EM SPA) has advanced three-dimensional protein structure determination, yet resolving intrinsically disordered proteins and regions (IDPs/IDRs) remains challenging due to conformational heterogeneity. This research evaluates cryo-EM’s capacity to map dynamic regions, assesses the adaptability of disorder prediction tools, and explores optimization strategies for dynamic structure prediction. Cryo-EM SPA datasets from 2000 to 2024 were categorized into different periods, forming a database integrating sequence data and disorder indices. Established prediction tools—AlphaFold2 (pLDDT), flDPnn, and IUPred—were evaluated for transferability, while a multi-level CLTC hybrid model (combining CNN, LSTM, Transformer, and CRF architectures) was developed to link local conformational fluctuations with global sequence contexts. Analyses revealed consistent advancements in average resolution and model counts over the past decade, although mapping disordered regions remained technically demanding. Both the adapted AlphaFold pLDDT and the CLTC model demonstrated efficacy in predicting structurally variable and poorly resolved regions. A subset of the cryo-EM missing residues exhibited intermediate conformational features, suggesting classification ambiguities potentially influenced by experimental conditions. These findings systematically outline the evolving capabilities of cryo-EM in resolving dynamic regions, benchmark the adaptability of computational tools, and introduce a hybrid model to enhance prediction accuracy. This study provides a framework for addressing conformational heterogeneity, contributing to methodological advancements in structural biology. Full article

Organs-on-Chips (OoC) technology has begun to be considered a pragmatic tool for drug evaluation, offering researchers an opportunity to move beyond the less physiologically relevant animal models. OoCs are microfluidic structures that imitate the functionalities of individual human organs, serving as mimicry tools for drug response and reproducibility studies. On the one hand, companies producing OoCs find managing and analyzing the large amounts of data generated challenging. This is where artificial intelligence (AI) can be deployed to address such problems. This paper will present the state-of-the-art of current OoC technology and AI, discussing the benefits and threats of combining these approaches. AI can be applied to optimize the process of OoC fabrication and operation, as well as for the big data analysis of OoC devices. By combining these technologies, scientists gain a powerful tool for drug development that is more efficient and accurate. However, processing the vast datasets generated by OoC systems often requires specialized AI expertise and computational resources. Despite the numerous possible benefits of amalgamating OoC technology with AI, several challenges and limitations need to be addressed. The large datasets generated by OoC systems can be difficult to process and analyze, which is a task that may require specialized AI expertise. Additionally, limitations of OoC systems include issues with reproducibility, as the devices are sensitive to perturbations in experimental conditions. Furthermore, the development and implementation of AI algorithms require significant computational resources and expertise, which may not be readily available to all research institutions. To overcome these challenges, interdisciplinary collaboration between biologists, engineers, data scientists, and AI experts is essential. Continued advancements in both OoC technology and AI will likely lead to more robust and versatile platforms for biomedical research and drug development, ultimately contributing to the advancement of personalized medicine and the reduction of reliance on animal testing. Full article

Type 2 diabetes (T2D) is a chronic metabolic disorder requiring effective management to avoid complications. Metformin is a first-line drug agent and is routinely prescribed for the control of glycemia, but its underlying dynamics are complicated and not fully quantified. This paper formulates a control-oriented and interpretable mathematical model that integrates metformin dynamics into a classic beta-cell–insulin–glucose (BIG) regulation system. The paper’s applicability to theoretical and clinical settings is enhanced by rigorous mathematical analysis, which guarantees the model is globally bounded, well-posed, and biologically meaningful. One of the key features of the study is its global stability analysis using Lyapunov functions, which demonstrates the asymptotic stability of critical equilibrium points under realistic physiological constraints. These findings support the predictive reliability of the model in explaining long-term glycemic regulation. Bifurcation analysis also clarifies the dynamic interplay between glucose production and utilization by identifying parameter thresholds that signify transitions between homeostasis and pathological states. Residual analysis, which detects Gaussian-distributed errors, underlines the robustness of the fitting process and suggests possible refinements by including temporal effects. Sensitivity analysis highlights the predominant effect of the initial dose of metformin on long-term glucose regulation and provides practical guidance for optimizing individual treatment. Furthermore, changing the two considered metformin parameters from their optimal values—altering the dose by ±50% and the decay rate by ±20%—demonstrates the flexibility of the model in simulating glycemic responses, confirming its adaptability and its potential for optimizing personalized treatment strategies. Full article

The overexpression of atypical protein kinase C-iota (PKC-ι) is a biomarker for carcinogenesis in various cell types, such as glioma, ovarian, renal, etc., manifesting as a potential drug target. In previous in vitro studies, ICA-1S and ICA-1T, experimental candidates for inhibiting PKC-ι, have demonstrated their specificity and promising efficacy against various cancers. Moreover, the in vivo studies have demonstrated low toxicity levels in acute and chronic murine models. Despite these prior developments, the binding affinities of the inhibitors were never thoroughly explored from a biophysical perspective. Here, we present the biophysical characterizations of PKC-ι in combination with ICA-1S/1T. Various methods based on molecular docking, light scattering, intrinsic fluorescence, thermal denaturation, and heat exchange were applied. The biophysical characteristics including particle sizing, thermal unfolding, aggregation profiles, enthalpy, entropy, free energy changes, and binding affinity (K_d) of the PKC-ι in the presence of ICA-1S were observed. The studies indicate the presence of domain-specific stabilities in the protein–ligand complex. Moreover, the results indicate a spontaneous reaction with an entropic gain, resulting in a possible entropy-driven hydrophobic interaction and hydrogen bonds in the binding pocket. Altogether, these biophysical studies reveal important insights into the binding interactions of PKC-ι and its inhibitors ICA-1S/1T. Full article

Protein purification is often performed for various applications. However, enzyme purification processes typically involve multiple steps that reduce yield and increase production costs. To overcome these challenges, we developed a novel three-step process to purify a lipase from whole sardine viscera (WSV), leveraging protein properties and the structural affinity of lipases for dye ligands. A crude extract of the viscera (CEV) was obtained by grinding the whole viscera in 50 mM phosphate buffer (pH 7.0, Solution B) followed by centrifugation (6000× g; 30 min, 0 °C). Lipolytic activity (3.3 U/mg) was recorded only in the supernatant. The purification process began with ammonium sulfate fractionation (30–50% saturation), increasing lipolytic activity in the precipitate (PF30-50) to 32.9 U/mg. PF30-50 was then ultrafiltered using a 30 KDa MWCO membrane, where 5% of semi-purified lipases (SPLSV) was retained with an activity of 156.5 U/mg (UF30). Finally, the SPLSV was injected into a column packed with dye ligand affinity adsorbent, pre-equilibrated with 1.0 M ammonium sulfate in buffer A. The WSV lipase was eluted using a step gradient to progressively reduce salt concentration. SDS-PAGE analysis revealed a single band of purified lipase from sardine viscera (PLSV) corresponding to a molecular weight of 123.4 kDa, with a specific activity of 266.4 U/mg. The combination of ammonium sulfate precipitation, ultrafiltration, and dye-ligand affinity chromatography provides a scalable and reproducible approach with potential industrial relevance, particularly in biocatalysis and waste valorization contexts. Full article

Protein polarimetry has been evaluated as a simple and straightforward technique to detect the cryptic denaturation of exemplary proteins. The general rules of rotation vs. amino acid and structural composition and the respective knowledge gaps were reviewed, and the specific rotation of cystine was determined in 4 M NaCl solution as [α]_D²⁰ = –302.5°. The specific rotations at 589 nm and 436 nm and the ratio were measured for several model proteins, some purified plasma-derived proteins and for three monoclonal antibodies. The immunoglobulin G concentrates all showed a narrow ratio range likely characteristic for this protein class. Heat denaturation experiments were conducted at temperatures between 50 and 85 °C both for short-time (10 min) and for prolonged periods of heat exposure (up to 210 min). Denaturation by heat resulted not only in the known levorotatory shift, but also in a shift in the specific rotation ratio. The stabilizing effect of fatty acids in bovine serum could be demonstrated by this parameter. Polarimetry thus appears to be a particularly sensitive and simple method for the characterization of the identity and the thermal stability of proteins and should therefore be added again as a complimentary method to the toolbox of protein chemistry. Full article

Protein enzymes are highly efficient catalysts that exhibit adaptability and selectivity under diverse biological conditions. In some organisms, such as bacteria, structurally similar enzymes, for instance, shikimate kinase (SK) and adenylate kinase (AK), coexist and act on chemically related ligands. This raises the question of whether these enzymes can accommodate and potentially react with each other’s ligands. In this study, we investigate the stability of non-cognate ligand binding in SK and explore whether external electric fields (EFs) can modulate this interaction, leading to cross-reactivity in SK. Using molecular dynamics simulations, we assess the structural integrity of SK and the binding behavior of ATP and AMP under EF-off and EF-on cases. Our results show that EFs enhance protein structure stability, stabilize non-cognate ligands in the binding pocket, and reduce local energetic frustration near the R116 residue located in the binding site. In addition to this, dimensionality reduction analyses reveal that EFs induce more coherent protein motions and reduce the number of metastable states. Together, these findings suggest that external EFs can reshape enzyme–ligand interactions and may serve as a tool to modulate enzymatic specificity and functional promiscuity. Thus, we provide computational evidence that supports the concept of using an EF as a tunable parameter in enzyme engineering and synthetic biology. However, further experimental investigation would be valuable to assess the reliability of our computational predictions. Full article

Icariin (ICA) is widely recognized for its health benefits. In this work, we examined the intermolecular interactions between ICA and proteinase K (PK) via multi-spectroscopic techniques and molecular simulations. The experimental findings revealed that ICA quenched the fluorescence emission of PK by forming a noncovalent complex. Both hydrogen bonding and van der Waals interactions are essential for the complex’s formation. Then Förster resonance energy transfer (FRET), competitive experiments, and synchronous fluorescence spectroscopy were adopted to verify the formation of the complex. Molecular docking studies demonstrated that ICA could spontaneously bind to PK by hydrogen bonding and hydrophobic interactions, which is consistent with the spectroscopic results. The PK-ICA complex’s dynamic stability was evaluated using a 50 ns molecular dynamics (MD) simulation. The simulation results revealed no significant structural deformation or positional changes throughout the entire simulation period. The complex appears to be rather stable, as seen by the average root-mean-square deviation (RMSD) fluctuations for the host protein in the PK-ICA complex of 1.08 Å and 3.09 Å. These outcomes of molecular simulations suggest that ICA interacts spontaneously and tightly with PK, consistent with the spectroscopic findings. The approach employed in this research presents a pragmatic and advantageous method for examining protein–ligand interactions, as evidenced by the concordance between empirical and theoretical findings. Full article

This study extends the application of photothermal spectroscopy to explore heat transfer dynamics in biological fluids, focusing on the examination of artificial vitreous humor (VH) models of human VH and an endogenous sample of cervine (deer) VH. The research integrates previously established methods for analyzing thermal lensing through photothermal deflection. By visualizing convective and conductive heat transfer processes in the artificial components of human VH, one gains insights into the dynamic behavior of heat transfer in the VH. Relevance extends to clinical cases where pathology requires replacement of endogenous VH with an artificial VH substitute. Several VH substitutes identified in the literature were chosen for this study based on their physical properties and relative abundance in the VH. Individual component fluids, and mixtures of these components, were analyzed at various concentrations based on their physiological concentration ranges in the human VH as they varied with age, sex, and certain disease states. By way of comparison to endogenous biological VH, a sample of VH obtained from a female white-tailed deer eye was analyzed, enhancing the understanding of heat transfer in artificial components of the VH compared to endogenous VH. There is a vast array of ophthalmological procedures that utilize an external heat source interacting with endogenous or artificial VH. The data found in this study will progress the understanding of heat transfer within artificial VH components in comparison to endogenous VH and contribute to the advancement of certain ophthalmological procedures. Full article

Recent major advancements in cryo-electron microscopy (cryo-EM) have enabled high-resolution structural analysis, accompanied by developments in image processing software packages for single-particle analysis (SPA). SPA facilitates the 3D reconstruction of proteins and macromolecular complexes from numerous individual particles. In this study, we systematically evaluated the impact of symmetry parameters and particle quantity on the 3D reconstruction efficiency using the dihydrolipoyl acetyltransferase (E2) inner core of the pyruvate dehydrogenase complex (PDC). We specifically examined how inappropriate symmetry constraints can introduce structural artifacts and distortions, underscoring the necessity for accurate symmetry determination through rigorous validation methods such as directional Fourier shell correlation (FSC) and local-resolution mapping. Additionally, our analysis demonstrates that efficient reconstructions can be achieved with a moderate particle number, significantly reducing computational costs without compromising structural accuracy. We further contextualize these results by discussing recent developments in SPA workflows and hardware optimization, highlighting their roles in enhancing reconstruction accuracy and computational efficiency. Overall, our comprehensive benchmarking provides strategic insights that will facilitate the optimization of SPA experiments, particularly in resource-limited settings, and offers practical guidelines for accurately determining symmetry and particle quantity during cryo-EM data processing. Full article

This study represents a first step toward improving the repeatability, reproducibility, and accuracy of a process designed to enhance dynamic water structuring. We aim is to investigate the optical reflectivity of a watery magnesium chloride solution treated with electromagnetic waves, we employ a novel methodology derived from human plethysmography (PPG) with three wavelengths spanning the visible and infrared spectra. We measured the reflectance of 17 flasks at 536 nm, 660 nm, and 940 nm before and after treatment, first using the succussion method (control) and second using a 50 Hz signal. The observed variability was acceptable, with repeatability errors below 0.15% and reproducibility errors below 3.5% across all wavelengths before and after treatment. Out of 51 samples dynamically structured using the succussion method, we obtained two false negatives, while one false negative was recorded out of 51 samples dynamically structured using the electromagnetic (EM) method. PPG appears to be a relevant sensor, as it correctly detected dynamically structured water in 99 out of 102 cases, using either the succussion or electromagnetic method. Our results show significant differences in reflectance (supposedly correlated with water’s structured status) at 536 nm between dynamically structured and dynamic non-structured samples (p < 0.001). Future improvements will include a validation protocol against gold-standard spectrophotometry with a larger sample size. Full article

We present a corrosion-resistant quadrupole ion trap mass spectrometer (QIT-MS) designed for trace detection of volatiles in sulfuric acid aerosols, with a specific focus on phosphine (PH₃). Here, we detail the gas calibration methodology using permeation tube technology for generating certified ppb-level PH₃/H₂S/CO₂ mixtures, and report results from mass spectra with sufficient resolution to distinguish isotopic envelopes that validate the detection of PH₃ at a concentration of 62 ppb. Fragmentation patterns for PH₃ and H₂S agree with NIST data, and signal-to-noise performance confirms ppb sensitivity over 2.6 h acquisition periods. We further assess spectral interferences from oxygen isotopes and propose a detection scheme based on isolated phosphorus ions (P⁺) to enable specific and interference-resistant identification of PH₃ and other reduced phosphorus species of astrobiological interest in Venus-like environments. This work extends the capabilities of QIT-MS for trace gas analysis in chemically aggressive atmospheric conditions. Full article

Coumarins are known for interacting with proteins and exhibiting diverse biological activities. This study investigates the interaction between coumarin-343 (C343) and human (HSA) and bovine (BSA) serum albumins. Fluorescence spectroscopy and theoretical simulations, including density functional theory (DFT) and molecular docking, were used to analyze the ligand–protein complex formation. The fluorescence quenching data revealed that C343 binds to both proteins, with binding constants of 2.1 × 10⁵ mol·L⁻¹ (HSA) and 6.5 × 10⁵ mol·L⁻¹ (BSA), following a 1:1 stoichiometry. Binding site markers identified drug site I (DS1), located in subdomain IIA, as the preferential binding region for both proteins. Computational results supported these findings, showing high affinity for DS1, with binding energies of −69.02 kcal·mol⁻¹ (HSA) and −67.22 kcal·mol⁻¹ (BSA). While complex formation was confirmed for both proteins, differences emerged in the induced circular dichroism (ICD) signals. HSA displayed a distinct ICD profile compared to BSA in both intensity and absorption maximum. Molecular Docking revealed that the C343 conformation differed between HSA and BSA, explaining the variation in ICD signals. These results highlight the importance of protein structure in modulating ligand interactions and spectral responses. Full article

An engineered molecular motor composed of an ATP-dependent kinesin-1 monomer and an ATP-independent diffusing microtubule-associated protein is proposed, and its dynamics are studied theoretically. It is shown that the engineered motor can move directionally on microtubules towards the plus end, bearing great potential for applications in therapeutics or nanorobotics. The engineered motor can have an unloaded velocity similar to the wild-type kinesin-1 dimer, can take a mechanical (either forward or backward) step by hydrolyzing an ATP molecule under any load, and can generate the maximum force that is about half of that generated by the wild-type kinesin-1 dimer. Full article

A brief summary of the effect of nonspecific interactions upon chemical equilibria in solutions containing a high total concentration of macromolecular solutes comparable to that found in biological fluid media is presented. Analyses of experimental measurements permitting relatively direct quantitation of the free energy of nonspecific intermolecular interaction in solutions of one or two macrosolutes are described, and a table listing published experimental studies of both homo- and hetero-interactions is provided. Methods for calculating the free energy of nonspecific interaction via theory and computer simulation are described. Recommendations for further progress in both measurement and calculation of interaction free energies are presented. Full article

Collagen II is a vital structural component in developing bones and mature cartilage. Mutations in this protein cause spondyloepiphyseal dysplasia, a disease characterized primarily by altered skeletal growth and manifesting with a range of phenotypes, from lethal to mild. This study examined transgenic mice harboring the R992C (p.R1124C) substitution in collagen II. Previous research demonstrated significant growth abnormalities and disorganized growth plate structure in these mice, and histological signs of osteoarthritic changes in the knee joints of 9-month-old mice with the R992C mutation. Our study focuses on detecting early structural changes in the articular cartilage that occur before histological signs become apparent. Through microscopic and spectroscopic analyses, we observed significant alterations in the distribution gradients of collagenous proteins and proteoglycans in the cartilage of R992C mutant mice. We propose that these early changes, eventually leading to articular cartilage degeneration in older mice, underscore the progressive nature of osteoarthritic changes linked to collagen II mutations. By identifying these early structural aberrations, our findings emphasize the importance of early detection of osteoarthritic changes, potentially facilitating timely, non-surgical interventions. Full article

An ancient repertoire of ultraviolet (UV)-absorbing pigments which survive today in the phylogenetically oldest extant photosynthetic organisms, the cyanobacteria, point to a direction in evolutionary adaptation of the pigments and their associated biota; from largely UV-C absorbing pigments in the Archean to pigments covering ever more of the longer wavelength UV and visible regions in the Phanerozoic. Since photoprotection is not dependent on absorption, such a scenario could imply selection of photon dissipation rather than photoprotection over the evolutionary history of life, consistent with the thermodynamic dissipation theory of the origin and evolution of life which suggests that the most important hallmark of biological evolution has been the covering of Earth’s surface with organic pigment molecules and water to absorb and dissipate ever more completely the prevailing surface solar spectrum. In this article we compare a set of photophysical, photochemical, biosynthetic, and other inherent properties of the two dominant classes of cyanobacterial UV-absorbing pigments, the mycosporine-like amino acids (MAAs) and scytonemins. We show that the many anomalies and paradoxes related to these biological pigments, for example, their exudation into the environment, spectral coverage of the entire high-energy part of surface solar spectrum, their little or null photoprotective effect, their origination at UV-C wavelengths and then spreading to cover the prevailing Earth surface solar spectrum, can be better understood once photodissipation, and not photosynthesis or photoprotection, is considered as being the important variable optimized by nature. Full article

Ark shells are a group of bivalves that exhibit extraordinary adaptability to the dual environmental pressures of low oxygen and osmotic imbalance. These challenges are particularly pronounced in intertidal zones, where organisms are subjected to rapid and drastic changes in their surroundings. This research investigated the molecular mechanisms that underpin their survival and adaptive strategies, with particular focused on sodium–potassium ATPase (NKA), a pivotal enzyme responsible for maintaining cellular ion transmembrane gradients and ensuring cellular homeostasis under stress conditions. By utilizing genome assemblies and transcriptomics datasets from multiple ark shell species, we successfully identified two distinct NKA-α subunits and two NKA-β subunits, which are essential components of the NKA complex. Moreover, the discovery of a conserved NKA-interacting protein (NKAIN) highlights the complexity and evolutionary significance of the NKA-NKAIN system in ark shells. Phylogenetic analysis revealed a high degree of conservation in the NKA-α and NKA-β subunits across ark shells, suggesting strong selective pressures to preserve their functionality. However, the marked divergence observed between the two NKA-β subunits suggests that they may serve distinct roles in ion transport, potentially specialized for specific environmental conditions or stress responses. Comparative transcriptomic analysis further revealed the regulatory roles of NKA and NKAIN in the adaptive responses to hypoxia and osmotic stress, showing that these genes are dynamically modulated at the transcriptional level in response to environmental challenges. These findings provide a molecular foundation for understanding the osmotic adaptation mechanisms in ark shells and offer novel insights into their ability to thrive in mudflat habitats. This comprehensive exploration of the NKA-NKAIN system not only enhances our understanding of the resilience of ark shells but also provides valuable insights into the molecular and physiological strategies employed by bivalves in intertidal environments. Full article

The system suitability testing of chromatography is an indispensable procedure in pharmaceutical analysis, and it must comply with rules in related pharmacopoeias. An inverse Fourier transform algorithm was developed to accurately evaluate chromatographic features versus a standard Gaussian peak shape. The regular chromatogram is considered a pseudo-frequency spectrum and can be converted to a nominal time signal via inverse Fourier transformation. The system suitability parameters of peak width, theoretical plate number, tailing factor, and noise testing were evaluated using linear regressions directly and compared with the compendial rules. This novel method is simple, accurate, robust, reliable, and efficient for the evaluation of chromatographic peak features. Full article