Biophysical reports最新文献

{"title":"Single-photon smFRET. III. Application to pulsed illumination","authors":"Matthew Safar, A. Saurabh, Bidyut Sarkar, M. Fazel, Kunihiko Ishii, T. Tahara, Ioannis Sgouralis, S. Pressé","doi":"10.1101/2022.07.20.500892","DOIUrl":"https://doi.org/10.1101/2022.07.20.500892","url":null,"abstract":"Förster resonance energy transfer (FRET) using pulsed illumination has been pivotal in leveraging lifetime information in FRET analysis. However, there remain major challenges in quantitative single photon, single molecule FRET (smFRET) data analysis under pulsed illumination including: 1) simultaneously deducing kinetics and number of system states; 2) providing uncertainties over estimates, particularly uncertainty over the number of system states; 3) taking into account detector noise sources such as crosstalk, and the instrument response function contributing to uncertainty; in addition to 4) other experimental noise sources such as background. Here, we implement the Bayesian nonparametric framework described in the first companion manuscript that addresses all aforementioned issues in smFRET data analysis specialized for the case of pulsed illumination. Furthermore, we apply our method to both synthetic as well as experimental data acquired using Holliday junctions. Why It Matters In the first companion manuscript of this series, we developed new methods to analyze noisy smFRET data. These methods eliminate the requirement of a priori specifying the dimensionality of the physical model describing a molecular complex’s kinetics. Here, we apply these methods to experimentally obtained datasets with samples illuminated by laser pulses at regular time intervals. In particular, we study conformational dynamics of Holliday junctions.","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"17 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76290200","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":"Transport between im/mobile fractions shapes the speed and profile of cargo distribution in neurons.","authors":"Adriano A Bellotti,&nbsp;Jonathan G Murphy,&nbsp;Timothy S O'Leary,&nbsp;Dax A Hoffman","doi":"10.1016/j.bpr.2022.100082","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100082","url":null,"abstract":"<p><p>Neuronal function requires continuous distribution of ion channels and other proteins throughout large cell morphologies. Protein distribution is complicated by immobilization of freely diffusing subunits such as on lipid rafts or in postsynaptic densities. Here, we infer rates of immobilization for the voltage-gated potassium channel Kv4.2. Fluorescence recovery after photobleaching quantifies protein diffusion kinetics, typically reported as a recovery rate and mobile fraction. We show that, implicit in the fluorescence recovery, are rates of particle transfer between mobile and immobile fractions (im/mobilization). We performed photobleaching of fluorescein-tagged ion channel Kv4.2-sGFP2 in over 450 dendrites of rat hippocampal cells. Using mass-action models, we infer rates of Kv4.2-sGFP2 im/mobilization. Using a realistic neuron morphology, we show how these rates shape the speed and profile of subunit distribution. The experimental protocol and model inference introduced here is widely applicable to other cargo and experimental systems.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100082"},"PeriodicalIF":0.0,"publicationDate":"2022-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/cf/1e/main.PMC9680811.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40484759","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":"Permissive and nonpermissive channel closings in CFTR revealed by a factor graph inference algorithm.","authors":"Alexander S Moffett,&nbsp;Guiying Cui,&nbsp;Peter J Thomas,&nbsp;William D Hunt,&nbsp;Nael A McCarty,&nbsp;Ryan S Westafer,&nbsp;Andrew W Eckford","doi":"10.1016/j.bpr.2022.100083","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100083","url":null,"abstract":"<p><p>The closing of the gated ion channel in the cystic fibrosis transmembrane conductance regulator can be categorized as nonpermissive to reopening, which involves the unbinding of ADP or ATP, or permissive, which does not. Identifying the type of closing is of interest as interactions with nucleotides can be affected in mutants or by introducing agonists. However, all closings are electrically silent and difficult to differentiate. For single-channel patch-clamp traces, we show that the type of the closing can be accurately determined by an inference algorithm implemented on a factor graph, which we demonstrate using both simulated and lab-obtained patch-clamp traces.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100083"},"PeriodicalIF":0.0,"publicationDate":"2022-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9680790/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40486155","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":"Electrically stimulated droplet injector for reduced sample consumption in serial crystallography.","authors":"Mukul Sonker, Diandra Doppler, Ana Egatz-Gomez, Sahba Zaare, Mohammad T Rabbani, Abhik Manna, Jorvani Cruz Villarreal, Garrett Nelson, Gihan K Ketawala, Konstantinos Karpos, Roberto C Alvarez, Reza Nazari, Darren Thifault, Rebecca Jernigan, Dominik Oberthür, Huijong Han, Raymond Sierra, Mark S Hunter, Alexander Batyuk, Christopher J Kupitz, Robert E Sublett, Frederic Poitevin, Stella Lisova, Valerio Mariani, Alexandra Tolstikova, Sebastien Boutet, Marc Messerschmidt, J Domingo Meza-Aguilar, Raimund Fromme, Jose M Martin-Garcia, Sabine Botha, Petra Fromme, Thomas D Grant, Richard A Kirian, Alexandra Ros","doi":"10.1016/j.bpr.2022.100081","DOIUrl":"10.1016/j.bpr.2022.100081","url":null,"abstract":"<p><p>With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its \"off-time\" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"2 4","pages":"100081"},"PeriodicalIF":2.4,"publicationDate":"2022-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/48/eb/main.PMC9680787.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10565696","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":"Deciphering the conformational dynamics of gephyrin-mediated collybistin activation.","authors":"Nasir Imam,&nbsp;Susobhan Choudhury,&nbsp;Katherina Hemmen,&nbsp;Katrin G Heinze,&nbsp;Hermann Schindelin","doi":"10.1016/j.bpr.2022.100079","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100079","url":null,"abstract":"<p><p>Efficient neuronal signaling depends on the proper assembly of the postsynaptic neurotransmitter machinery. The majority of inhibitory synapses feature γ-aminobutyric acid type A (GABA_A) receptors. The function of these GABAergic synapses is controlled by the scaffolding protein gephyrin and collybistin, a Dbl family guanine nucleotide exchange factor and neuronal adaptor protein. Specifically, collybistin interacts with small GTPases, cell adhesion proteins, and phosphoinositides to recruit gephyrin and GABA_A receptors to postsynaptic membrane specializations. Collybistin usually contains an N-terminal SH3 domain and exists in closed/inactive or open/active states. Here, we elucidate the molecular basis of the gephyrin-collybistin interaction with newly designed collybistin Förster resonance energy transfer (FRET) sensors. Using fluorescence lifetime-based FRET measurements, we deduce the affinity of the gephyrin-collybistin complex, thereby confirming that the C-terminal dimer-forming E domain binds collybistin, an interaction that does not require E domain dimerization. Simulations based on fluorescence lifetime and sensor distance distributions reveal at least a two-state equilibrium of the SH3 domain already in the free/unbound collybistin, thereby illustrating the accessible volume of the SH3 domain. Finally, our data provide strong evidence for a tightly regulated collybistin-gephyrin interplay, where, unexpectedly, switching of collybistin from closed/inactive to open/active states is efficiently triggered by gephyrin.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100079"},"PeriodicalIF":0.0,"publicationDate":"2022-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f8/6c/main.PMC9680708.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40486156","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":"A comparative study of interfacial environments in lipid nanodiscs and vesicles.","authors":"Xiao You,&nbsp;Naveen Thakur,&nbsp;Arka Prabha Ray,&nbsp;Matthew T Eddy,&nbsp;Carlos R Baiz","doi":"10.1016/j.bpr.2022.100066","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100066","url":null,"abstract":"<p><p>Membrane protein conformations and dynamics are driven by the protein-lipid interactions occurring within the local environment of the membrane. These environments remain challenging to accurately capture in structural and biophysical experiments using bilayers. Consequently, there is an increasing need for realistic cell-membrane mimetics for <i>in vitro</i> studies. Lipid nanodiscs provide certain advantages over vesicles for membrane protein studies. Nanodiscs are increasingly used for structural and spectroscopic characterization of membrane proteins. Despite the common use of nanodiscs, the interfacial environments of lipids confined to a ~10-nm diameter area have remained relatively underexplored. Here, we use ultrafast two-dimensional infrared spectroscopy and temperature-dependent infrared absorption measurements of the ester carbonyls to compare the interfacial hydrogen bond structure and dynamics in lipid nanodiscs of varying lipid compositions and sizes with ~100-nm vesicles. We examine the effects of lipid composition and nanodisc size. We found that nanodiscs and vesicles share largely similar lipid-water H-bond environments and interfacial dynamics. Differences in measured enthalpies of H-bonding suggest that H-bond dynamics in nanodiscs are modulated by the interaction between the annular lipids and the scaffold protein.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"2 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d4/19/main.PMC9518727.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9623325","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":"Identification and quantification of within-burst dynamics in singly labeled single-molecule fluorescence lifetime experiments.","authors":"Paul David Harris,&nbsp;Eitan Lerner","doi":"10.1016/j.bpr.2022.100071","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100071","url":null,"abstract":"<p><p>Single-molecule spectroscopy has revolutionized molecular biophysics and provided means to probe how structural moieties within biomolecules spatially reorganize at different timescales. There are several single-molecule methodologies that probe local structural dynamics in the vicinity of a single dye-labeled residue, which rely on fluorescence lifetimes as readout. Nevertheless, an analytical framework to quantify dynamics in such single-molecule single dye fluorescence bursts, at timescales of microseconds to milliseconds, has not yet been demonstrated. Here, we suggest an analytical framework for identifying and quantifying within-burst lifetime-based dynamics, such as conformational dynamics recorded in single-molecule photo-isomerization-related fluorescence enhancement. After testing the capabilities of the analysis on simulations, we proceed to exhibit within-burst millisecond local structural dynamics in the unbound <i>α</i>-synuclein monomer. The analytical framework provided in this work paves the way for extracting a full picture of the energy landscape for the coordinate probed by fluorescence lifetime-based single-molecule measurements.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/1b/01/main.PMC9534301.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33510528","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":"Heterogeneous migration routes of DNA triplet repeat slip-outs.","authors":"Simona Bianco,&nbsp;Tianyu Hu,&nbsp;Oliver Henrich,&nbsp;Steven W Magennis","doi":"10.1016/j.bpr.2022.100070","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100070","url":null,"abstract":"<p><p>It is unclear how the length of a repetitive DNA tract determines the onset and progression of repeat expansion diseases, but the dynamics of secondary DNA structures formed by repeat sequences are believed to play an important role. It was recently shown that three-way DNA junctions containing slip-out hairpins of CAG or CTG repeats and contiguous triplet repeats in the adjacent duplex displayed single-molecule FRET (smFRET) dynamics that were ascribed to both local conformational motions and longer-range branch migration. Here we explore these so-called \"mobile\" slip-out structures through a detailed kinetic analysis of smFRET trajectories and coarse-grained modeling. Despite the apparent structural simplicity, with six FRET states resolvable, most smFRET states displayed biexponential dwell-time distributions, attributed to structural heterogeneity and overlapping FRET states. Coarse-grained modeling for a (GAC)₁₀ repeat slip-out included trajectories that corresponded to a complete round of branch migration; the structured free energy landscape between slippage events supports the dynamical complexity observed by smFRET. A hairpin slip-out with 40 CAG repeats, which is above the repeat length required for disease in several triplet repeat disorders, displayed smFRET dwell times that were on average double those of 3WJs with 10 repeats. The rate of secondary-structure rearrangement via branch migration, relative to particular DNA processing pathways, may be an important factor in the expansion of triplet repeat expansion diseases.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"None"},"PeriodicalIF":0.0,"publicationDate":"2022-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9586884/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40665358","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":"Optimization of cryo-electron microscopy for quantitative analysis of lipid bilayers","authors":"Frederick A. Heberle, Doug Welsch, H. L. Scott, M. Waxham","doi":"10.1101/2022.08.23.505005","DOIUrl":"https://doi.org/10.1101/2022.08.23.505005","url":null,"abstract":"Cryogenic electron microscopy (cryo-EM) is among the most powerful tools available for interrogating nanoscale structure of biological structures. We recently showed that cryo-EM can be used to measure the bilayer thickness of lipid vesicles and biological membranes with sub-angstrom precision, resulting in the direct visualization of nanoscopic domains of different thickness in multicomponent lipid mixtures and giant plasma membrane vesicles. Despite the great potential of cryo-EM for revealing the lateral organization of biomembranes, a large parameter space of experimental conditions remains to be optimized. Here, we systematically investigate the influence of instrument parameters and image post-processing steps on the ability to accurately measure bilayer thickness and discriminate regions of different thickness within unilamellar liposomes. We also demonstrate a spatial autocorrelation analysis to extract additional information about lateral heterogeneity. Significance Raft domains in unstimulated cells have proven difficult to directly visualize owing to their nanoscopic size and fleeting existence. The few techniques capable of nanoscopic spatial resolution typically rely on interpretation of indirect spectroscopic or scattering signals or require stabilizing the membrane on a solid support. In contrast, cryo-EM yields direct images of nanoscale domains in probe-free, unsupported membranes. Here, we systematically optimize key steps in the experimental and analysis workflow for this new and specialized application. Our findings represent an important step toward developing cryo-EM into a robust method for investigating phase behavior of membranes at length scales relevant to lipid rafts.","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"183 12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78579660","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":"Is orange carotenoid protein photoactivation a single-photon process?","authors":"Stanisław Niziński,&nbsp;Ilme Schlichting,&nbsp;Jacques-Philippe Colletier,&nbsp;Diana Kirilovsky,&nbsp;Gotard Burdzinski,&nbsp;Michel Sliwa","doi":"10.1016/j.bpr.2022.100072","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100072","url":null,"abstract":"<p><p>In all published photoactivation mechanisms of orange carotenoid protein (OCP), absorption of a single photon by the orange dark state starts a cascade of red-shifted OCP ground-state intermediates that subsequently decay within hundreds of milliseconds, resulting in the formation of the final red form OCP^R, which is the biologically active form that plays a key role in cyanobacteria photoprotection. A major challenge in deducing the photoactivation mechanism is to create a uniform description explaining both single-pulse excitation experiments, involving single-photon absorption, and continuous light irradiation experiments, where the red-shifted OCP intermediate species may undergo re-excitation. We thus investigated photoactivation of <i>Synechocystis</i> OCP using stationary irradiation light with a biologically relevant photon flux density coupled with nanosecond laser pulse excitation. The kinetics of photoactivation upon continuous and nanosecond pulse irradiation light show that the OCP^R formation quantum yield increases with photon flux density; thus, a simple single-photon model cannot describe the data recorded for OCP <i>in vitro</i>. The results strongly suggest a consecutive absorption of two photons involving a red intermediate with ≈100 millisecond lifetime. This intermediate is required in the photoactivation mechanism and formation of the red active form OCP^R.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100072"},"PeriodicalIF":0.0,"publicationDate":"2022-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/3a/12/main.PMC9680785.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40704942","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}