Biophysical journal最新文献

{"title":"Advances in FRET methodologies for probing molecular interactions.","authors":"Geetika Verma,Vasanthi Jayaraman","doi":"10.1016/j.bpj.2025.08.027","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.08.027","url":null,"abstract":"Förster resonance energy transfer (FRET) has evolved into a powerful, quantitative approach for probing biomolecular structure, dynamics, and interactions. This research highlight brings together recent studies in Biophysical Journal that push the boundaries of traditional FRET. These innovations expand the spatial and temporal resolution of FRET, enabling its application to increasingly complex biological systems.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"33 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145127156","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biophysical Mechanisms of SARS-CoV-2-Induced Surfactant Inhibition.","authors":"Guangle Li,Xiaojie Xu,Bingbing Sun,Yi Y Zuo","doi":"10.1016/j.bpj.2025.09.028","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.09.028","url":null,"abstract":"Surfactant replacement has been studied as a supportive therapy for managing COVID-19-induced acute respiratory distress syndrome (ARDS). The clinical applications require biophysical understanding of the molecular mechanisms behind SARS-CoV-2-induced surfactant inhibition. While SARS-CoV-2 is known to attack alveolar type II epithelial cells, it is unknown whether the virus can directly interact with the pulmonary surfactant film adsorbed at the alveolar surface. The virus utilizes its spike (S) protein, consisting of two functional subunits (S1 and S2), to bind to the host cell membrane and mediate subsequent membrane fusion. We hypothesize that these two subunits may differentially interact with pulmonary surfactant, resulting in distinct effects on surfactant inhibition. The biophysical impact of recombinant S1 and S2 subunit proteins on a bovine-extracted natural pulmonary surfactant film was investigated with combined constrained drop surfactometry and atomic force microscopy. Our findings revealed that the S2 subunit, in contrast to the S1 subunit, selectively induces surfactant inhibition, evidenced by its capacity in reducing dynamic surface activity and causing domain fusion in surfactant monolayers. These results contribute novel insights into the biophysical mechanisms underlying surfactant inhibition in SARS-CoV-2-induced ARDS, and may hold translational implications for advancing surfactant therapy to manage COVID-19.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"35 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145103538","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Predicting Side Chain Conformations in Folded Proteins by AlphaFold: Perspective and Challenges.","authors":"Gia G Maisuradze,Abhishek Thakur,Kisan Khatri,Allan Haldane,Ronald M Levy","doi":"10.1016/j.bpj.2025.09.030","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.09.030","url":null,"abstract":"AlphaFold has revolutionized protein structure prediction by accurately creating 3D structures from just the amino-acid sequence. However, a key question important for the molecular modeling field remains: Can AlphaFold predict the conformations of individual amino-acid residue side chains within a folded protein? Herein, we investigate the ability of ColabFold, an online implementation of AlphaFold2, and AlphaFold3 to predict the side-chain conformations in folded proteins. We find that over a set of 10 benchmark proteins (set A) representing several different highly-populated fold families, which are included in the AlphaFold protein structure database, the side-chain conformation prediction error of ColabFold is ∼14% for χ1 dihedral angles, and increases to ∼48% for χ3 dihedral angles. Prediction error is smaller for non-polar side chains and is somewhat improved using structural templates. ColabFold demonstrates a bias towards the most prevalent rotamer states in protein data bank, potentially limiting its ability to capture rare side-chain conformations effectively. Additionally, for 10 recently-released protein structures, which were not employed in the training of AlphaFold2, we show that ColabFold predicts side-chain conformations with almost the same accuracy as for the set A. Also, we demonstrate the side-chain prediction accuracy by AlphaFold3 is slightly better than by ColabFold. As an application of AlphaFold to explore the structural consequences of strongly cooperative mutations on side-chain rearrangements, we employ a Potts sequence-based statistical energy model to perform large scale mutational scans of two proteins ABL1 and PIM1 kinase, searching for the most strongly cooperative mutational pairs, and then use ColabFold to predict the structural signatures of this cooperativity on the interacting side chains. Our results demonstrate that integration of the sequence-based Potts model with AlphaFold into a single pipeline provides a new tool that can be used to explore the fundamental relationship between protein mutations, and cooperative changes in structure, and fitness.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"56 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145103537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reactive oxygen species counteract zebrafish wound contraction and promote wound healing.","authors":"Chang Ding, Linlin Li, Yueyang Wang, Hong-Anh A Nguyen, Deva D Chan, David M Umulis, Adrian T Buganza, Qing Deng","doi":"10.1016/j.bpj.2025.09.027","DOIUrl":"10.1016/j.bpj.2025.09.027","url":null,"abstract":"<p><p>Reactive oxygen species (ROS) are second messengers that drive wound closure. However, the mechanism by which ROS regulate wound contraction to facilitate wound healing remains unclear. Here, we report that ROS counteract wound contraction by inhibiting the phosphorylation of myosin regulatory light chain. Acute ROS inhibition, through pharmacological perturbations, disturbs wound relaxation, delays wound closure, and impairs regrowth after amputation. Moreover, actomyosin inhibition relaxes tailfin contraction without impairing wound closure or regrowth. Overcontraction, on the other hand, impedes wound closure. Meanwhile, chronic depletion of epithelial ROS during embryonic development, achieved through morpholino-mediated knockdown of the duox gene, alters tissue stiffness, as measured using atomic force microscopy-based nanoindentation. Despite a reduced contraction force, the wound also appears to be overcontracted, with delayed healing and regrowth. An in silico linear elasticity simulation to calculate the second principal stress based on node-wise prescribed displacement recapitulated the contraction dynamics during acute and chronic ROS inhibition. Together, our results provide a novel understanding of how ROS facilitate wound closure, a process instrumental in restoring tissue integrity and maintaining homeostasis.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145102602","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spatiotemporal dynamics of local anesthetic diffusion in nerve revealed by a 2D computational model.","authors":"Vladimir Smrkolj,Jakob Kralj,Janez Mavri,Nejc Umek","doi":"10.1016/j.bpj.2025.09.026","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.09.026","url":null,"abstract":"Despite their extensive clinical use, the intra-neural pharmacokinetics of local anesthetics, including the mechanisms determining their onset and duration, remain incompletely understood, particularly under pathological conditions. We developed a detailed, spatially-compartmentalized computational model of human peripheral nerve fascicles to simulate the diffusion, accumulation, and clearance kinetics of lidocaine and bupivacaine under physiological and acidic conditions. The model integrates nerve fiber architecture, extracellular fluid compartments, and capillary-mediated clearance, parameterized using experimentally validated anatomical and physicochemical data. It was implemented in Python 3.12, employing a fourth-order Runge-Kutta integrator via the SciPy library. The results revealed that onset is limited primarily by extracellular diffusion rather than transmembrane transport. Lidocaine reached the predefined onset threshold (≥50% of nerve fibers with ≥50% external concentration) at 8.7 s under physiological pH and 3.2 s under acidic conditions. Bupivacaine exhibited longer onset times, 79.2 s and 16.1 s, respectively. Acidic conditions markedly reduced equilibrium concentrations within nerve fibers (by 3.6-fold for lidocaine, 3.5-fold for bupivacaine), significantly shortening their durations of action (lidocaine: 758 to 233 s; bupivacaine: 6980 to 2059 s). These findings mirror known clinical efficacy reductions in inflamed or acidotic tissues. In conclusion, local anesthetic onset is primarily governed by extracellular diffusion rather than membrane permeability, rendering it largely independent of drug pKa. Local tissue acidosis reduces nerve fiber accumulation and accelerates anesthetic washout, thereby diminishing both efficacy and duration of action. These results explain longstanding clinical observations and suggest that nerve fibers act as kinetic reservoirs modulated by tissue pH. This computational model offers a valuable framework for predicting anesthetic behavior and optimizing drug delivery strategies in regional anesthesia, particularly under pathological conditions such as inflammation.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"18 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145089746","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single-Molecule FRET and Tracking of Transfected Biomolecules in Living Cells.","authors":"Abhinaya Anandamurugan,Antonia Eidloth,Veronika Frank,Philipp Wortmann,Lukas Schrangl,Chenyang Lan,Gerhard J Schütz,Thorsten Hugel","doi":"10.1016/j.bpj.2025.09.024","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.09.024","url":null,"abstract":"Proteins and DNA in cells exhibit different conformational states, which are influenced by dynamic interactions with other biomolecules. All these interactions are affected by the molecules' localization within the cell, i.e., their compartmentalization. Such, in cellula, compartment-specific dynamics is difficult to measure, because of limitations in instrumentation, autofluorescence of cells, and the necessity to track diffusing molecules. Here, we present a bottom-up engineering approach, which allows us to track transfected proteins in cellula and to analyze time-resolved single-molecule FRET efficiencies. This has been achieved by alternating laser excitation (ALEX) based dual-channel (donor and FRET, acceptor) tracking with a HILO microscope. We validate our strategy by characterizing long-term static FRET traces of customized DNA with known dye positions. We utilize two different transfection strategies, namely Microinjection (physical) and a transfection mediated by the toxin Streptolysin-O (biological). By comparing in vitro and in cellula measurements we show that the cellular environment in this case changes the FRET efficiency by about 25%. In addition, we evaluate single-molecule FRET traces for the heat shock protein Hsp90 in cellula. The obtained FRET efficiency distribution is largely consistent with known Hsp90 structures and in vitro distributions, but also shows some clear differences. Altogether, we show that FRET-TTB (Förster Resonance Energy Transfer-Tracking of Transfected Biomolecules) opens the path to study protein state changes of transfected biomolecules in living cells, including their time-resolved cellular localization.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"1 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145089747","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single VLP lipid-mixing measurements confirm off-pathway state in dengue virus fusion mechanism.","authors":"Tasnim K Anika, Fiona Campbell, Bianca Linden, Connor J Criswell, Alice Walker, Miranda Kimm, Priscilla Li-Ning Yang, Robert J Rawle","doi":"10.1016/j.bpj.2025.09.025","DOIUrl":"10.1016/j.bpj.2025.09.025","url":null,"abstract":"<p><p>Dengue virus (DENV) is the causative agent of dengue fever and exerts a substantial healthcare burden worldwide. Like other flaviviruses, DENV must undergo membrane fusion with the host cell in order to initiate infection. This membrane fusion occurs after acidification during endocytosis and is pH dependent. Here, we interrogate whether the mechanism of DENV fusion contains an off-pathway state, such has been reported previously for two other flaviviruses-Zika virus and West Nile virus. To do this, we utilize single-particle lipid-mixing measurements of DENV virus-like particles (VLPs) to tethered liposomes, together with computational modeling inspired by chemical kinetics. By observing and then modeling the pH dependence of single-VLP fusion kinetics, we provide evidence that the DENV fusion mechanism must contain an off-pathway state. Measuring the proportion of VLPs undergoing hemi-fusion over time, we also demonstrate that the off-pathway state appears to be slowly reversible over tens of minutes, at least for some virions. Additionally, we find that late endosomal anionic lipids do not appear to influence the off-pathway mechanism to any great extent. In conjunction with the prior reports on Zika virus and West Nile virus, this work indicates that an off-pathway fusion state may be a feature of flavivirus fusion more broadly. We also note that the platform and mechanistic model described in this study may be useful in elucidating the mechanism of action of small molecule inhibitors of flavivirus fusion developed by our group and others.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145091038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Chromatin Compaction Scaling with Cell Size Follows a Power Law from Interphase Through Mitosis.","authors":"Petra Stockinger,Anna Oddone,Melike Lakadamyali,Manuel Mendoza,Jérôme Solon","doi":"10.1016/j.bpj.2025.09.021","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.09.021","url":null,"abstract":"Coordination of mitotic chromosome compaction with cell size is crucial for proper genome segregation during mitosis. During development, DNA content remains constant but cell size changes dynamically, necessitating a mechanism that scales chromosome compaction with cell size to ensure proper chromatin segregation. In this study, we examined chromatin compaction in the developing Drosophila nervous system by analyzing the large neuronal stem cells and their smaller progeny, the ganglion mother cells. Using super-resolution 3D Stochastic Optical Reconstruction Microscopy and quantitative time-lapse fluorescence microscopy, we observed that nanoscale chromatin density during interphase scales with nuclear volume according to a power law. This scaling relationship is disrupted by inhibiting histone deacetylase activity, indicating that molecular cues rather than mechanical constraints primarily regulate chromatin compaction. Notably, this power law dependency is maintained into mitosis but the scaling exponent decreases. This suggests a phase separation-like transition in the biophysical state of chromatin, whereby the polymer shifts from a more expanded to a more compact configuration. Accordingly, we propose that the scaling of mitotic chromosome size relative to cell size emerges from the organisational principles of interphase chromatin, and that mitotic compaction may be governed by polymer properties modulated by changes in the chromatin - solvent environment.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"68 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145083357","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spatial Charge-Hydrophobicity Configuration Modulates Cationic Peptide Transport in Cartilage.","authors":"Bill Hakim,Timothy L Boyer,Srirupa Chakraborty,Ambika G Bajpayee","doi":"10.1016/j.bpj.2025.09.023","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.09.023","url":null,"abstract":"Charge-based delivery systems offer a promising approach for targeting dense, negatively charged tissues such as cartilage, which presents a significant transport barrier due to its high fixed charge density (FCD) from aggrecan glycosaminoglycans (GAGs). Cationic nano-carriers, including peptide-based systems, can overcome these barriers by leveraging electrostatic interactions to enhance intra-tissue penetration. However, the effectiveness of these carriers depends not only on their net positive charge, which drives Donnan partitioning, but also on the precise spatial arrangement of cationic and hydrophobic residues, which influences transport, binding, and retention. In this study, we investigated the impact of spatial charge distribution and hydrophobicity on the intra-cartilage transport and retention of arginine-rich cationic peptide carriers (CPCs) with a net charge of +14, optimized for effective cartilage targeting. Using both experimental methods and molecular modeling, we examined the transport properties of CPCs with varied charge and hydrophobic cluster arrangements in healthy and degenerated cartilage with different FCDs. Our findings reveal that peptides with a higher degree of clustered cationic or hydrophobic residues exhibit greater intra-cartilage diffusivity due to weaker binding interactions with aggrecan GAGs and a more flexible structural conformation that incurs an entropic penalty. However, while hydrophobic residues can enhance intra-tissue retention, particularly in degenerated tissues, they also promote competitive binding within synovial fluid (SF), emphasizing the need for hydrophilic designs. Overall, our results indicate that evenly distributed cationic residues and minimal hydrophobicity yield the most effective carriers for deep, long-term tissue penetration, providing a framework for the rational design of tissue-targeting cationic peptide carriers. The design principles established in this work can be broadly applied to the rational development of cationic carriers for targeted drug delivery in a wide range of negatively charged tissues.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"105 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145083402","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Markovian state models uncover casein kinase 1 dynamics that govern circadian period.","authors":"Clarisse Gravina Ricci, Jonathan M Philpott, Megan R Torgrimson, Alfred M Freeberg, Rajesh Narasimamurthy, Emilia Pécora de Barros, Rommie Amaro, David M Virshup, J Andrew McCammon, Carrie L Partch","doi":"10.1016/j.bpj.2025.09.022","DOIUrl":"10.1016/j.bpj.2025.09.022","url":null,"abstract":"<p><p>Circadian rhythms in mammals are tightly regulated through phosphorylation of period (PER) proteins by casein kinase 1 (CK1, subtypes δ and ε). CK1 acts on at least two different regions of PER with opposing effects: phosphorylation of phosphodegron regions leads to PER degradation, whereas phosphorylation of the familial advanced sleep phase (FASP) region leads to PER stabilization. To investigate how substrate selectivity is encoded by the conformational dynamics of CK1, we performed a large set of independent molecular dynamics simulations of wild-type CK1 and the tau mutant (R178C) that biases kinase activity toward a phosphodegron. We used Markovian state models to integrate the simulations into a single model of the conformational landscape of CK1 and used Gaussian accelerated molecular dynamics to build the first molecular model of CK1 and the unphosphorylated FASP motif. These findings were biochemically validated using in vitro kinase assays and provide a mechanistic view of CK1, establishing how the activation loop acts as a key molecular switch to control substrate selectivity. We show that the wild-type CK1 prefers a \"loop down\" conformation that binds FASP, whereas the tau mutant favors an alternative conformation of the activation loop and significantly accelerates the dynamics of CK1. This reshapes the binding cleft in a way that impairs FASP binding and would ultimately lead to PER destabilization. Finally, we identified a potential binding pocket that could be targeted to influence the conformational state of this molecular switch and lead to predictable changes in circadian period. Our integrated approach offers a detailed model of CK1's conformational landscape and its relevance to normal, mutant, and druggable circadian timekeeping.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145085076","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}