Biophysical reports最新文献

{"title":"Structural studies of the human α₁ glycine receptor via site-specific chemical cross-linking coupled with mass spectrometry.","authors":"Rathna J Veeramachaneni, Chelsee A Donelan, Kayce A Tomcho, Shaili Aggarwal, David J Lapinsky, Michael Cascio","doi":"10.1016/j.bpr.2024.100184","DOIUrl":"10.1016/j.bpr.2024.100184","url":null,"abstract":"<p><p>By identifying distance constraints, chemical cross-linking coupled with mass spectrometry (CX-MS) can be a powerful complementary technique to other structural methods by interrogating macromolecular protein complexes under native-like conditions. In this study, we developed a CX-MS approach to identify the sites of chemical cross-linking from a single targeted location within the human α1 glycine receptor (α1 GlyR) in its apo state. The human α1 GlyR belongs to the family of pentameric ligand-gated ion channel receptors that function in fast neurotransmission. A single chemically reactive cysteine was reintroduced into a Cys null α1 GlyR construct at position 41 within the extracellular domain of human α1 homomeric GlyR overexpressed in a baculoviral system. After purification and reconstitution into vesicles, methanethiosulfonate-benzophenone-alkyne, a heterotrifunctional cross-linker, was site specifically attached to Cys41 via disulfide bond formation. The resting receptor was then subjected to UV photocross-linking. Afterward, monomeric and oligomeric α1 GlyR bands from SDS-PAGE gels were trypsinized and analyzed by tandem MS in bottom-up studies. Dozens of intrasubunit and intersubunit sites of α1 GlyR cross-linking were differentiated and identified from single gel bands of purified protein, showing the utility of this experimental approach to identify a diverse array of distance constraints of the α1 GlyR in its resting state. These studies highlight CX-MS as an experimental approach to identify chemical cross-links within full-length integral membrane protein assemblies in a native-like lipid environment.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100184"},"PeriodicalIF":2.7,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11550363/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142407301","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expression level of cardiac ryanodine receptors dictates properties of Ca²⁺-induced Ca²⁺ release.","authors":"Roman Nikolaienko, Elisa Bovo, Aleksey V Zima","doi":"10.1016/j.bpr.2024.100183","DOIUrl":"10.1016/j.bpr.2024.100183","url":null,"abstract":"<p><p>The type 2 ryanodine receptor (RyR2) is the major Ca²⁺ release channel required for Ca²⁺-induced Ca²⁺ release (CICR) and cardiac excitation-contraction coupling. The cluster organization of RyR2 at the dyad is critical for efficient CICR. Despite its central role in cardiac Ca²⁺ signaling, the mechanisms that control CICR are not fully understood. As a single RyR2 Ca²⁺ flux dictates local CICR that underlies Ca²⁺ sparks, RyR2 density in a cluster, and therefore the distance between RyR2s, should have a profound impact on local CICR. Here, we studied the effect of the RyR2 expression level ([RyR2]) on CICR activation, termination, and amplitude. The endoplasmic reticulum (ER)-targeted Ca²⁺ sensor RCEPIA-1er was used to directly measure the ER [Ca²⁺] (Ca²⁺]_ER) in the T-Rex-293 the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) stable cell line expressing human RyR2. Cells coexpressing RyR2 and SERCA2a produced periodic [Ca²⁺]_ER depletions in the form of spontaneous Ca²⁺ waves due to propagating CICR. For each studied cell, the [Ca²⁺]_ER at which Ca²⁺ waves are activated and terminated was analyzed as a function of [RyR2]. CICR parameters, such as [Ca²⁺]_ER activation, termination, and amplitude, were inversely proportional to [RyR2] at low-intermediate levels. Increasing the sensitivity of RyR2 to cytosolic Ca²⁺ lowered the [Ca²⁺]_ER at which CICR is activated and terminated. Decreasing the sensitivity of RyR2 to cytosolic Ca²⁺ had the opposite effect on CICR. These results suggest that RyR2 density in the release cluster should have a significant impact on local CICR activation and termination. Since SR Ca²⁺ load is evenly distributed throughout the SR network, clusters with higher RyR2 density would have a higher probability of initiating spontaneous CICR.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100183"},"PeriodicalIF":2.4,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11532243/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142333654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a digital amplifier system for cut-open oocyte electrophysiology.","authors":"Lucas J Koerner, Ian Delgadillo Bonequi, Ian S K Shogren, Abraham Stroschein, Jordan Haag, Linda M Boland","doi":"10.1016/j.bpr.2024.100185","DOIUrl":"10.1016/j.bpr.2024.100185","url":null,"abstract":"<p><p>The cut-open oocyte Vaseline gap technique is a powerful electrophysiological method for the characterization of ion channels. However, traditional amplifiers for cut-open oocyte Vaseline gap are labor intensive and require significant user expertise. We introduce an innovative, open-source digital amplifier system with high-speed digitization and software-controlled electronics for computer-driven automation. This system compares well to existing commercial systems in terms of conventional specifications of step response (current peak at 25μs and decay of 36μs time constant), current noise (1.0 nA at 3-kHz bandwidth), and dynamic range (96.9 dB). Additionally, it unlocks new methods through close integration of the amplifier and software, including machine-learning techniques for tuning capacitive compensation waveforms, achieving a 100-fold suppression of mean-squared transient current, and impedance measurement methods to identify system components such as membrane capacitance and electrode resistances. For future extensions, the design has unique attributes such as real-time digital signal processing for feedback, multiple input and multiple output, and allows for user customization. By providing open-source access to the circuit board designs, control software, and field-programmable gate array code on GitHub, this approach aims to foster cross-disciplinary collaboration and facilitate instrument customization enabling previously inaccessible electrophysiology experiments.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100185"},"PeriodicalIF":2.7,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11549981/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142482224","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An effective drift-diffusion model for pandemic propagation and uncertainty prediction.","authors":"Clara Bender, Abhimanyu Ghosh, Hamed Vakili, Preetam Ghosh, Avik W Ghosh","doi":"10.1016/j.bpr.2024.100182","DOIUrl":"10.1016/j.bpr.2024.100182","url":null,"abstract":"<p><p>Predicting pandemic evolution involves complex modeling challenges, typically involving detailed discrete mathematics executed on large volumes of epidemiological data. Making them physics based provides added intuition as well as predictive value. Differential equations have the advantage of offering smooth, well-behaved solutions that try to capture overall predictive trends and averages. In this paper, the canonical susceptible-infected-recovered model is simplified, in the process generating quasi-analytical solutions and fitting functions that agree well with the numerics, as well as infection data across multiple countries. The equations provide an elegant way to visualize the evolution of the pandemic spread, by drawing equivalents with the similar dynamics of a particle, whose location over time represents the growing fraction of the population that is infected. This particle slides down a potential whose shape is set by model epidemiological parameters such as reproduction rate. Potential sources of errors and their growth over time are identified, and the uncertainties are mapped into a diffusive jitter that tends to push the particle away from its minimum. The combined physical understanding and analytical expressions offered by such an intuitive drift-diffusion model sets the foundation for their eventual extension to a multi-patch model while offering practical error bounds and could thus be useful in making policy decisions going forward.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100182"},"PeriodicalIF":2.4,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11775906/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142302323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering stress as a motivation for filamentous virus morphology.","authors":"Andrew McMahon, Swetha Vijayakrishnan, Hafez El Sayyed, Danielle Groves, Michaela J Conley, Edward Hutchinson, Nicole C Robb","doi":"10.1016/j.bpr.2024.100181","DOIUrl":"10.1016/j.bpr.2024.100181","url":null,"abstract":"<p><p>Many viruses are pleomorphic in shape and size, with pleomorphism often thought to correlate with infectivity, pathogenicity, or virus survival. For example, influenza and respiratory syncytial virus particles range in size from small spherical virions to filaments reaching many micrometers in length. We have used a pressure vessel model to investigate how the length and width of spherical and filamentous virions can vary for a given critical stress and fluorescence super-resolution microscopy along with image analysis tools to fit imaged influenza viruses to the model. We have shown that influenza virion dimensions fit within the theoretical limits of the model, suggesting that filament formation may be a way to increase an individual virus's volume without particle rupture. We have also used cryoelectron microscopy to investigate influenza and respiratory syncytial virus dimensions at the extrema of the model and used the pressure vessel model to explain the lack of alternative virus particle geometries. Our approach offers insight into the possible purpose of filamentous virus morphology and is applicable to a wide range of other biological entities, including bacteria and fungi.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100181"},"PeriodicalIF":2.4,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11447354/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142302324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Thank you to our reviewers.","authors":"","doi":"10.1016/j.bpr.2024.100188","DOIUrl":"https://doi.org/10.1016/j.bpr.2024.100188","url":null,"abstract":"","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"4 4","pages":"100188"},"PeriodicalIF":2.4,"publicationDate":"2024-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142796658","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Magnetic field platform for experiments on well-mixed and spatially structured microbial populations.","authors":"Akila Bandara, Enoki Li, Daniel A Charlebois","doi":"10.1016/j.bpr.2024.100165","DOIUrl":"10.1016/j.bpr.2024.100165","url":null,"abstract":"<p><p>Magnetic fields have been shown to affect sensing, migration, and navigation in living organisms. However, the effects of magnetic fields on microorganisms largely remain to be elucidated. We develop an open-source, 3D-printed magnetic field exposure device to perform experiments on well-mixed and spatially structured microbial populations. This device is designed in AutoCAD, modeled in COMSOL, and validated using a Gaussmeter and experiments on the budding yeast Saccharomyces cerevisiae. We find that static magnetic field exposure slows the spatially structured expansion of yeast mats that expand in two dimensions, but not yeast mats that expand in three dimensions, across the surface of semi-solid yeast extract-peptone-dextrose agar media. We also find that magnetic fields do not affect the growth of planktonic yeast cells in well-mixed liquid yeast extract-peptone-dextrose media. This study provides an adaptable device for performing controlled magnetic field experiments on microbes and advances our understanding of the effects of magnetic fields on fungi.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100165"},"PeriodicalIF":2.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11276921/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141428435","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advanced analysis of single-molecule spectroscopic data.","authors":"Joshua L Botha, Bertus van Heerden, Tjaart P J Krüger","doi":"10.1016/j.bpr.2024.100173","DOIUrl":"10.1016/j.bpr.2024.100173","url":null,"abstract":"<p><p>We present Full SMS, a multipurpose graphical user interface (GUI)-based software package for analyzing single-molecule spectroscopy (SMS) data. SMS typically delivers multiparameter data-such as fluorescence brightness, lifetime, and spectra-of molecular- or nanometer-scale particles such as single dye molecules, quantum dots, or fluorescently labeled biological macromolecules. Full SMS allows an unbiased statistical analysis of fluorescence brightness through level resolution and clustering, analysis of fluorescence lifetimes through decay fitting, as well as the calculation of second-order correlation functions and the display of fluorescence spectra and raster-scan images. Additional features include extensive data filtering options, a custom HDF5-based file format, and flexible data export options. The software is open source and written in Python but GUI based so it may be used without any programming knowledge. A multiprocess architecture was employed for computational efficiency. The software is also designed to be easily extendable to include additional import data types and analysis capabilities.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100173"},"PeriodicalIF":2.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11374972/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141891106","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Lipid loss and compositional change during preparation of simple two-component liposomes.","authors":"Eunice Kim, Olivia Graceffa, Rachel Broweleit, Ali Ladha, Andrew Boies, Sanyukta Prakash Mudakannavar, Robert J Rawle","doi":"10.1016/j.bpr.2024.100174","DOIUrl":"10.1016/j.bpr.2024.100174","url":null,"abstract":"<p><p>Liposomes are used as model membranes in many scientific fields. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures can produce liposomes at specific concentrations and lipid compositions, and researchers often assume that the concentration and composition of their liposomes are similar or identical to what would be expected if no lipid loss occurred. However, lipid loss and concomitant biasing of lipid composition can in principle occur at any preparation step due to nonideal mixing, lipid-surface interactions, etc. Here, we report a straightforward HPLC-ELSD method to quantify the lipid concentration and composition of liposomes and apply that method to study the preparation of simple cholesterol/POPC liposomes. We examine common liposome preparation steps, including vortexing during resuspension, lipid film hydration, extrusion, freeze-thaw, and sonication. We found that the resuspension step can play an outsized role in determining the lipid loss (up to ∼50% under seemingly rigorous procedures). The extrusion step yielded smaller lipid losses (∼10-20%). Freeze-thaw and sonication could both be employed to improve lipid yields. Hydration times up to 60 min and increasing cholesterol concentrations up to 50 mol % had little influence on lipid recovery. Fortunately, even conditions with large lipid loss did not substantially influence the target membrane composition, as long as the lipid mixture was below the cholesterol solubility limit. From our results, we identify best practices for producing maximum levels of lipid recovery and minimal changes to lipid composition during liposome preparation for cholesterol/POPC liposomes.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100174"},"PeriodicalIF":2.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11406089/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142037898","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Numerical model for electrogenic transport by the ATP-dependent potassium pump KdpFABC.","authors":"Adel Hussein, Xihui Zhang, David L Stokes","doi":"10.1016/j.bpr.2024.100169","DOIUrl":"10.1016/j.bpr.2024.100169","url":null,"abstract":"<p><p>In vitro assays of ion transport are an essential tool for understanding molecular mechanisms associated with ATP-dependent pumps. Because ion transport is generally electrogenic, principles of electrophysiology are applicable, but conventional tools like patch-clamp are ineffective due to relatively low turnover rates of the pumps. Instead, assays have been developed to measure either voltage or current generated by transport activity of a population of molecules either in cell-derived membrane fragments or after reconstituting purified protein into proteoliposomes. In order to understand the nuances of these assays and to characterize effects of various operational parameters, we have developed a numerical model to simulate data produced by two relevant assays: fluorescence from voltage-sensitive dyes and current recorded by capacitive coupling on solid supported membranes. Parameters of the model, which has been implemented in Python, are described along with underlying principles of the computational algorithm. Experimental data from KdpFABC, a K⁺ pump associated with P-type ATPases, are presented, and model parameters have been adjusted to mimic these data. In addition, effects of key parameters such as nonselective leak conductance and turnover rate are demonstrated. Finally, simulated data are used to illustrate the effects of capacitive coupling on measured current and to compare alternative methods for quantification of raw data.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100169"},"PeriodicalIF":2.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11304011/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141478089","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}