Biophysical journal最新文献

{"title":"A closed-loop system for millisecond readout and control of membrane tension.","authors":"Michael Sindoni, Jörg Grandl","doi":"10.1016/j.bpj.2025.03.025","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.025","url":null,"abstract":"<p><p>Characterizing the function of force-gated ion channels is essential for understanding their molecular mechanisms and how they are affected by disease-causing mutations, lipids, or small molecules. Pressure-clamp electrophysiology is a method that is established and widely used to characterize the mechanical sensitivity of force-gated ion channels. However, the physical stimulus many force-gated ion channels sense is not pressure, but membrane tension. Here, we further develop the approach of combining patch-clamp electrophysiology with differential interference contrast microscopy into a system that controls membrane tension in real time. The system uses machine learning object detection for millisecond analysis of membrane curvature and control of pipette pressure to produce a closed-loop membrane tension clamp. The analysis of membrane tension is fully automated and includes propagation of experimental errors, thereby increasing throughput and reducing bias. A dynamic control program clamps membrane tension with at least 93% accuracy and 0.3 mN/m precision. Additionally, the absence of tension drift enables averaging open probabilities of ion channels with low expression and/or unitary conductance over long durations. Using this system, we apply a tension step protocol and show that TMEM63A responds to tension with a tension of half-maximal activation of T₅₀ = 5.5±0.1 mN/m. Overall, this system allows for precise and efficient generation of tension-response relationships of force-gated ion channels.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143750834","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":"A Discard-and-Restart MD algorithm for the sampling of protein intermediate states.","authors":"Alan Ianeselli, Jonathon Howard, Mark B Gerstein","doi":"10.1016/j.bpj.2025.03.024","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.024","url":null,"abstract":"<p><p>We introduce a Discard-and-Restart molecular dynamics (MD) algorithm tailored for the sampling of realistic protein intermediate states. It aids computational structure-based drug discovery by reducing the simulation times to compute a \"quick sketch\" of folding pathways by up to 2000x. The algorithm iteratively performs short MD simulations and measures their proximity to a target state via a collective variable (CV) loss, which can be defined in a flexible fashion, locally or globally. Using the loss, if the trajectory proceeds toward the target, the MD simulation continues. Otherwise, it is discarded, and a new MD simulation is restarted, with new initial velocities randomly drawn from a Maxwell-Boltzmann distribution. The discard-and-restart algorithm demonstrates efficacy and atomistic accuracy in capturing the folding pathways in several contexts: (1) fast-folding small protein domains; (2) the folding intermediate of the prion protein PrP; and (3) the spontaneous partial unfolding of α-Tubulin, a crucial event for microtubule severing. During each iteration of the algorithm, we can perform AI-based analysis of the transitory conformations to find potential binding pockets, which could represent druggable sites. Overall, our algorithm enables systematic and computationally efficient exploration of conformational landscapes, enhancing the design of ligands targeting dynamic protein states.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742016","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":"Perspectives: Comparison of Deep Learning Segmentation Models on Biophysical and Biomedical Data.","authors":"J Shepard Bryan, Pedro Pessoa, Meysam Tavakoli, Steve Pressé","doi":"10.1016/j.bpj.2025.03.023","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.023","url":null,"abstract":"<p><p>Deep learning based approaches are now widely used across biophysics to help automate a variety of tasks including image segmentation, feature selection, and deconvolution. However, the presence of multiple competing deep learning architectures, each with its own advantages and disadvantages, makes it challenging to select an architecture best suited for a specific application. As such, we present a comprehensive comparison of common models. Here, we focus on the task of segmentation assuming typical (often small) training dataset sizes available from biophysics experiments and compare the following four commonly used architectures: convolutional neural networks, U-Nets, vision transformers, and vision state space models. In doing so, we establish criteria for determining optimal conditions under which each model excels, thereby offering practical guidelines for researchers and practitioners in the field.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143750756","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":"Charge distribution and helicity tune the binding of septin's amphipathic helix domain to membranes.","authors":"Christopher J Edelmaier, Stephen J Klawa, S Mahsa Mofidi, Qunzhao Wang, Shreeya Bhonge, Ellysa J D Vogt, Brandy N Curtis, Wenzheng Shi, Sonya M Hanson, Daphne Klotsa, M Gregory Forest, Amy S Gladfelter, Ronit Freeman, Ehssan Nazockdast","doi":"10.1016/j.bpj.2025.03.008","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.008","url":null,"abstract":"<p><p>Amphipathic helices (AHs) are secondary structures that can facilitate binding of proteins to the membrane by folding into a helix with hydrophobic and hydrophilic faces that interact with the same surfaces in the lipid membrane. Septins are cytoskeletal proteins that preferentially bind to domains of micron-scale curvature on the cell membrane. Studies have shown that AH domains in septin are essential for curvature sensing. We present the first computational study of septin AH interactions with lipid bilayers. Using all-atom simulations and metadynamics-enhanced sampling, we study the effect of charge distribution at the flanking ends of septin AH on the energy for helical folding and its consequences on the binding configuration and affinity to the membrane. This is relevant to septins, since the net positive charge on the flanking C-terminal amino acids is a conserved property across several organisms. Simulations revealed that the energy barrier for folding in the neutral-capped AH is much larger than the charge-capped AH, leading to a small fraction of AH folding and integration to the membrane compared to a significantly folded configuration in the bound charge-capped AH. These observations are consistent with the binding measurements of synthetic AH constructs with variable helicity to lipid vesicles. Additionally, we examined an extended AH sequence including eight amino acids upstream and downstream of the AH to mimic the native protein. Again, simulations and experiments show that the extended peptide, with a net positive charge at C-terminus, adopts a strong helical configuration in solution, giving rise to a higher membrane affinity. Altogether, these results identify the energy cost for folding of AHs as a regulator of AH binding configuration and affinity and provide a basic template for parameterizing AH-membrane interactions as a starting point for the future multiscale simulations for septin-membrane interactions.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143778882","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":"Flow Sensitive Ion Channels in Vascular Endothelial Cells: Mechanisms of Activation and Roles in Mechanotransduction.","authors":"Katie M Beverley, Sang Joon Ahn, Irena Levitan","doi":"10.1016/j.bpj.2025.03.021","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.021","url":null,"abstract":"<p><p>The purpose of this review is to evaluate the current knowledge about the mechanisms by which mechanosensitive ion channels are activated by fluid shear stress in endothelial cells. We focus on three classes of endothelial ion channels that are most well studied for their sensitivity to flow and roles in mechanotransduction: inwardly-rectifying K⁺ channels, Piezo channels and TRPV channels. We also discuss the mechanisms by which these channels initiate and contribute to mechanosensitive signaling pathways. Three types of mechanisms have been described for flow-induced activation of ion channels: 1) through interaction with apical membrane flow sensors, such as glycocalyx, which is likely to be deformed by flow, 2) directly by sensing membrane stretch that is induced by shear stress, or 3) via flow-sensitive channel-channel or lipid channel interactions. We also demonstrate the physiological role of these channels and how they are related to cardiovascular and neurological diseases. Further studies are needed to determine how these channels function cooperatively to mediate the endothelial response to flow.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742018","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":"Agent-based model of the human colon to investigate mechanisms of pathogen colonization resistance.","authors":"Samantha Johanna Fletcher, Carly Ching, Mark Paladin Suprenant, Darash Desai, Muhammad Hamid Zaman","doi":"10.1016/j.bpj.2025.03.022","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.022","url":null,"abstract":"<p><p>Recent global burden of disease studies have shown that bacterial infections are responsible for over 13 million deaths worldwide, or one in every eight deaths, each year. Enteric diarrheal infections, in particular, pose a significant challenge and strain on healthcare systems as many are difficult to address pharmaceutically, and thus, rely primarily on the patient's own immune system and gut microbiome to fight the infection. Nonetheless, the specific mechanisms behind gut microbiome colonization resistance of enteric pathogens are not well-defined and microbiome diversity is difficult to represent and study experimentally. To address this gap, we have constructed an agent-based computational model of the colonic epithelium cross-section to investigate the colonic invasion of enteric pathogens. The model focuses on three main regions: epithelial layer, mucosal bilayer, and adjacent lumen, and utilizes four main cell types as agents: anaerobic bacteria, facultative anaerobic bacteria, human goblet cells, and pathogens. Utilizing this model, we are able to describe the healthy microbiome cell-localization and dynamics from our mucosal bilayer. In addition, we are also able to investigate the impact of host dietary fiber consumption and simulate pathogen invasion. The model exemplifies the possibility and potential to explore key gut microbiome colonization resistance mechanisms and environmental impacts on the gut microbiome using computational methods.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742017","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":"A novel \"bio-tag\" for cryo-EM studies based on the small, electron-dense protein Csp1","authors":"Weekie Yao, Adam C. Oken, David L. Farrens","doi":"10.1016/j.bpj.2025.03.018","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.018","url":null,"abstract":"Small proteins can be challenging to study by single-particle cryogenic electron microscopy (cryo-EM) techniques because they have low signal-to-noise ratios, making them difficult to identify and analyze. Here we investigated the use of Csp1, a small (54 kDa) tetrameric metal-binding protein, to act as a “bio-tag” to help overcome this problem. We find Csp1 is compact, stable, and exhibits enhanced electron scattering and excellent particle contrast in cryo-EM micrographs. As a result, we could determine the structure of Csp1 to 2.98-Å resolution using standard cryo-EM approaches. We also tested if Csp1 could be used as a tag or fiducial to help determine the structure of a protein bound to it. Specifically, we analyzed an epitope-tagged Csp1 bound to a ∼40-kDa Fab fragment from the antibody 1D4. Data from these complexes yielded medium-resolution structures of the complex (5.70 Å) and the bound 1D4 Fab (5.40 Å). These results suggest that, with further optimization, electron-rich Csp1 is a promising bio-tag for use in cryo-EM studies.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"12 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143823085","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":"Are cell length and volume interchangeable in cell cycle analysis?","authors":"Prathitha Kar, Ariel Amir","doi":"10.1016/j.bpj.2025.03.019","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.019","url":null,"abstract":"Cell length has been used as a proxy for cell size in cell cycle modeling studies. A previous study, however, brought into question the validity of this assumption, noting that correlations between cell lengths can be different from those involving cell volume if cell width fluctuations are taken into account. If cell volume is regulated, data analysis involving cell lengths will lead to an incorrect inference of the cell size control mechanism. We used conditional correlation of length variables conditioned upon radius variables to elucidate the underlying volume control mechanism. Using the conditional correlation on previous mother machine datasets measuring lengths at birth and division and the cell radius for multiple cells, we find that the cell volume control strategy is consistent with an adder model. Further, using the conditional correlation, we conclude that measurement noise constitutes a significant portion of the radius variability in the experimental datasets. To conclude, cell length and cell volume can often be used interchangeably owing to small cell width fluctuations.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"36 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143823084","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":"Reversible tuning of membrane sterol levels by cyclodextrin in a dialysis setting.","authors":"Cynthia Alsayyah, Emmanuel Rodrigues, Julia Hach, Mike F Renne, Robert Ernst","doi":"10.1016/j.bpj.2025.03.020","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.03.020","url":null,"abstract":"<p><p>Large unilamellar vesicles are popular membrane models for studying the impact of lipids and bilayer properties on the structure and function of transmembrane proteins. However, the functional reconstitution of transmembrane proteins in liposomes can be challenging, especially, if the hydrophobic thickness of the protein does not match the thickness of the lipid bilayer. Such hydrophobic mismatch causes protein aggregation and low yields during the reconstitution procedure, which are exacerbated in sterol-rich membranes featuring low membrane compressibility. Here, we explore new approaches to reversibly tune the sterol content of (proteo)liposomes with methyl-β-cyclodextrin (mβCD) in a dialysis setting. Maintaining (proteo)liposomes in a confined compartment minimizes loss-of-material during cholesterol transfer and facilitates efficient removal of mβCD. We monitor the sterol concentration in the membrane with help of the solvatochromic probe C-Laurdan, which reports on lipid packing. Using Förster-resonance energy transfer, we show that cholesterol delivery to proteoliposomes induces the oligomerization of a membrane property sensor, while a subsequent removal of cholesterol demonstrates full reversibility. We propose that tuning membrane compressibility by mβCD-meditated cholesterol delivery and removal in a dialysis setup provides a new handle to study the impact of sterols and membrane compressibility on membrane protein structure, function, and dynamics.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717901","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":"Mapping the distribution and affinities of ligand interaction sites on human serum albumin.","authors":"Asuka A Orr, Agbo-Oma Uwakweh, Xun Li, Ahmad Kiani Karanji, Stephen W Hoag, Daniel J Deredge, Alexander D MacKerell","doi":"10.1016/j.bpj.2025.03.016","DOIUrl":"10.1016/j.bpj.2025.03.016","url":null,"abstract":"<p><p>Ligands in many instances interact with a protein at multiple sites with a range of affinities. In this study, ligand-protein interaction sites on human serum albumin (HSA) are mapped using the site-identification by ligand competitive saturation (SILCS)-Biologics approach in conjunction with hydrogen-deuterium exchange (HDX)-mass spectrometry (MS) experiments. Ligands studied include known HSA binders, ibuprofen and ketoprofen, and compounds arginine, alanine, sucrose, and trehalose, excipients used in therapeutic formulations of protein-based drugs. In addition, the impact of excipient binding to HSA on its stability is investigated through temperature-ramp stability studies monitoring solution viscosity. For the studied ligands, interactions that correspond to known drug-binding sites (DSs) are identified. These include previously identified ibuprofen and ketoprofen interaction sites as well as additional sites and, in the case of the excipients, the ligands are shown to also bind at previously unidentified interaction sites, termed excipient sites (ESs) with 20 or more sites identified for the studied compounds. HDX-MS titrations were used to determine dissociation constants for a subset of the interaction sites for ibuprofen, ketoprofen, arginine, and sucrose, which exhibited K_d values in the low micromolar to millimolar range in satisfactory agreement with SILCS-Biologics predicted affinities, validating the computational approach to identify both high- and low-affinity interaction sites. The stability studies indicate the excipients offer protection at low excipient/protein ratios up to 66 with destabilization occurring at ratios above 132 with the exception of sucrose at the t₀ time point, indicating that the more favorable affinities of sucrose seen in the SILCS-Biologics and HDX-MS analyses contribute to protein stabilization. These results indicate that ligands can bind to large numbers of interaction sites on proteins, with those interactions having implications for the development of formulations for therapeutic proteins.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143708312","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}