Biophysical journal最新文献

{"title":"FTIR study of light-induced proton transfer and Ca²⁺ binding in T82D mutant of TAT rhodopsin.","authors":"Teppei Sugimoto, Kota Katayama, Hideki Kandori","doi":"10.1016/j.bpj.2024.08.005","DOIUrl":"10.1016/j.bpj.2024.08.005","url":null,"abstract":"<p><p>Proton transfer reactions play important functional roles in many proteins, such as enzymes and transporters, which is also the case in rhodopsins. In fact, functional expression of rhodopsins accompanies intramolecular proton transfer reactions in many cases. One of the exceptional cases can be seen in the protonated form of marine bacterial TAT rhodopsin, which isomerizes the retinal by light but returns to the original state within 10^-5 s. Thus, light energy is converted into heat without any function. In contrast, the T82D mutant of TAT rhodopsin conducts the light-induced deprotonation of the Schiff base at high pH. In this article, we report the structural analysis of T82D by means of difference Fourier transform infrared (FTIR) spectroscopy. In the light-induced difference FTIR spectra at 77 K, we observed little hydrogen out-of-plane vibrations for T82D as well as the wild-type (WT), suggesting that the planar chromophore structure itself is not the origin of the reversion from the K intermediate in WT TAT rhodopsin. Upon relaxation of the K intermediate, T82D forms the following intermediate, such as M, whereas K of WT returns to the original state. Present FTIR analysis revealed the proton transfer from the Schiff base to D82 in T82D upon formation of the M intermediate. It is accompanied by the second proton transfer from E54 to the Schiff base, forming the N intermediate, particularly in membranes. The equilibrium between the M and N intermediates corresponds to the protonation equilibrium between E54 and the Schiff base. We also found that Ca²⁺ binding takes place in T82D as well as WT but with 6 times lower affinity. An altered hydrogen-bonding network would be the origin of low affinity in T82D, where deprotonation of E54 is involved in the Ca²⁺ binding.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4245-4255"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141905801","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":"Nernst equilibrium, rectification, and saturation: Insights into ion channel behavior.","authors":"Ryan Carlsen, Hannah Weckel-Dahman, Jessica M J Swanson","doi":"10.1016/j.bpj.2024.10.016","DOIUrl":"10.1016/j.bpj.2024.10.016","url":null,"abstract":"<p><p>The dissipation of electrochemical gradients through ion channels plays a central role in biology. Herein we use voltage-responsive kinetic models of ion channels to explore how electrical and chemical potentials differentially influence ion transport properties. These models demonstrate how electrically driven flux is greater than the Nernstian equivalent chemically driven flux yet still perfectly cancels when the two gradients oppose each other. We find that the location and relative stability of ion-binding sites dictates rectification properties by shifting the location of the most voltage-sensitive transitions. However, these rectification properties invert when bulk concentrations increase relative to the binding-site stabilities, moving the rate-limiting steps from uptake into a relatively empty channel to release from an ion-blocked full channel. Additionally, the origin of channel saturation is shown to depend on the free energy of uptake relative to bulk concentrations. Collectively these insights provide a framework for interpreting and predicting how channel properties manifest in electrochemical transport behavior.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4304-4315"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142543339","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":"Dimerization is required for the glycosylation of S1-S2 linker of sea urchin voltage-gated proton channel Hv1.","authors":"Yoshifumi Okochi, Yuka Jinno, Yasushi Okamura","doi":"10.1016/j.bpj.2024.07.034","DOIUrl":"10.1016/j.bpj.2024.07.034","url":null,"abstract":"<p><p>Multimerization of ion channels is essential for establishing the ion-selective pathway and tuning the gating regulated by membrane potential, second messengers, and temperature. Voltage-gated proton channel, Hv1, consists of voltage-sensor domain and coiled-coil domain. Hv1 forms dimer, whereas voltage-dependent channel activity is self-contained in monomer unlike many ion channels, which assemble to form ion-conductive pathways among multiple subunits. Dimerization of Hv1 is necessary for cooperative gating, but other roles of dimerization in physiological aspects are still largely unclear. In this study, we show that dimerization of Hv1 takes place in ER. Sea urchin Hv1 (Strongylocentrotus purpuratus Hv1: SpHv1) was glycosylated in the consensus sequence for N-linked glycosylation within the S1-S2 extracellular loop. However, glycosylation was not observed in the monomeric SpHv1 that lacks the coiled-coil domain. A version of mHv1 in which the S1-S2 loop was replaced by that of SpHv1 showed glycosylation and its monomeric form was not glycosylated. Tandem dimer of monomeric SpHv1 underwent glycosylation, suggesting that dimerization of Hv1 is required for glycosylation. Moreover, when monomeric Hv1 has a dilysine motif in the C-terminal end, which is known to act as a retrieval signal from Golgi to ER, prolonging the time of residency in ER, it was glycosylated. Overall, our results suggest that monomeric SpHv1 does not stay long in ER, thereby escaping glycosylation, while the dimerization causes the proteins to stay longer in ER. Thus, the findings highlight the novel significance of dimerization of Hv1: regulation of biogenesis and maturation of the proteins in intracellular compartments.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4221-4232"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141858923","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":"Effects of pH on opioid receptor activation and implications for drug design.","authors":"Christoph Stein","doi":"10.1016/j.bpj.2024.07.007","DOIUrl":"10.1016/j.bpj.2024.07.007","url":null,"abstract":"<p><p>G-protein-coupled receptors are integral membrane proteins that transduce chemical signals from the extracellular matrix into the cell. Traditional drug design has considered ligand-receptor interactions only under normal conditions. However, studies on opioids indicate that such interactions are very different in diseased tissues. In such microenvironments, protons play an important role in structural and functional alterations of both ligands and receptors. The pertinent literature strongly suggests that future drug design should take these aspects into account in order to reduce adverse side effects while preserving desired effects of novel compounds.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4158-4166"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141544487","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":"Interaction with stomatin directs human proton channels into cholesterol-dependent membrane domains.","authors":"Artem G Ayuyan, Vladimir V Cherny, Gustavo Chaves, Boris Musset, Fredric S Cohen, Thomas E DeCoursey","doi":"10.1016/j.bpj.2024.03.003","DOIUrl":"10.1016/j.bpj.2024.03.003","url":null,"abstract":"<p><p>Many membrane proteins are modulated by cholesterol. Here we report profound effects of cholesterol depletion and restoration on the human voltage-gated proton channel, hH_V1, in excised patches but negligible effects in the whole-cell configuration. Despite the presence of a putative cholesterol-binding site, a CARC motif in hH_V1, mutation of this motif did not affect cholesterol effects. The murine H_V1 lacks a CARC sequence but displays similar cholesterol effects. These results argue against a direct effect of cholesterol on the H_V1 protein. However, the data are fully explainable if H_V1 preferentially associates with cholesterol-dependent lipid domains, or \"rafts.\" The rafts would be expected to concentrate in the membrane/glass interface and to be depleted from the electrically accessible patch membrane. This idea is supported by evidence that H_V1 channels can diffuse between seal and patch membranes when suction is applied. Simultaneous truncation of the large intracellular N and C termini of hH_V1 greatly attenuated the cholesterol effect, but C truncation alone did not; this suggests that the N terminus is the region of attachment to lipid domains. Searching for abundant raft-associated proteins led to stomatin. Co-immunoprecipitation experiment results were consistent with hH_V1 binding to stomatin. The stomatin-mediated association of H_V1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct H_V1 to subcellular locations where it is needed, such as the phagosome in leukocytes.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4180-4190"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140038629","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":"How and when to measure mitochondrial inner membrane potentials.","authors":"Alicia J Kowaltowski, Fernando Abdulkader","doi":"10.1016/j.bpj.2024.03.011","DOIUrl":"10.1016/j.bpj.2024.03.011","url":null,"abstract":"<p><p>The scientific literature on mitochondria has increased significantly over the years due to findings that these organelles have widespread roles in the onset and progression of pathological conditions such as metabolic disorders, neurodegenerative and cardiovascular diseases, inflammation, and cancer. Researchers have extensively explored how mitochondrial properties and functions are modified in different models, often using fluorescent inner mitochondrial membrane potential (ΔΨm) probes to assess functional mitochondrial aspects such as protonmotive force and oxidative phosphorylation. This review provides an overview of existing techniques to measure ΔpH and ΔΨm, highlighting their advantages, limitations, and applications. It discusses drawbacks of ΔΨm probes, especially when used without calibration, and conditions where alternative methods should replace ΔΨm measurements for the benefit of the specific scientific objectives entailed. Studies investigating mitochondria and their vast biological roles would be significantly advanced by the understanding of the correct applications as well as limitations of protonmotive force measurements and use of fluorescent ΔΨm probes, adopting more precise, artifact-free, sensitive, and quantitative measurements of mitochondrial functionality.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4150-4157"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140058620","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":"An affordable convertible: Engineering proton transfer pathways in microbial rhodopsins.","authors":"Leonid S Brown","doi":"10.1016/j.bpj.2024.08.014","DOIUrl":"10.1016/j.bpj.2024.08.014","url":null,"abstract":"","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4147-4149"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142035144","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":"Zinc inhibits the voltage-gated proton channel HCNL1.","authors":"Makoto F Kuwabara, Joschua Klemptner, Julia Muth, Emilia De Martino, Dominik Oliver, Thomas K Berger","doi":"10.1016/j.bpj.2024.08.018","DOIUrl":"10.1016/j.bpj.2024.08.018","url":null,"abstract":"<p><p>Voltage-gated ion channels allow ion flux across biological membranes in response to changes in the membrane potential. HCNL1 is a recently discovered voltage-gated ion channel that selectively conducts protons through its voltage-sensing domain (VSD), reminiscent of the well-studied depolarization-activated Hv1 proton channel. However, HCNL1 is activated by hyperpolarization, allowing the influx of protons, which leads to an intracellular acidification in zebrafish sperm. Zinc ions (Zn²⁺) are important cofactors in many proteins and essential for sperm physiology. Proton channels such as Hv1 and Otopetrin1 are inhibited by Zn²⁺. We investigated the effect of Zn²⁺ on heterologously expressed HCNL1 channels using electrophysiological and fluorometric techniques. Extracellular Zn²⁺ inhibits HCNL1 currents with an apparent half-maximal inhibition (IC₅₀) of 26 μM. Zn²⁺ slows voltage-dependent current kinetics, shifts the voltage-dependent activation to more negative potentials, and alters hyperpolarization-induced conformational changes of the voltage sensor. Our data suggest that extracellular Zn²⁺ inhibits HCNL1 currents by multiple mechanisms, including modulation of channel gating. Two histidine residues located at the extracellular side of the VSD might weakly contribute to Zn²⁺ coordination: mutants with either histidine replaced with alanine show modest shifts of the IC₅₀ values to higher concentrations. Interestingly, Zn²⁺ inhibits HCNL1 at even lower concentrations from the intracellular side (IC₅₀ ≈ 0.5 μM). A histidine residue at the intracellular end of S1 (position 50) is important for Zn²⁺ binding: much higher Zn²⁺ concentrations are required to inhibit the mutant HCNL1-H50A (IC₅₀ ≈ 106 μM). We anticipate that Zn²⁺ will be a useful ion to study the structure-function relationship of HCNL1 as well as the physiological role of HCNL1 in zebrafish sperm.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4256-4265"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142104008","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":"IP6, PF74 affect HIV-1 capsid stability through modulation of hexamer-hexamer tilt angle preference.","authors":"Chris M Garza, Matthew Holcomb, Diogo Santos-Martins, Bruce E Torbett, Stefano Forli","doi":"10.1016/j.bpj.2024.12.016","DOIUrl":"10.1016/j.bpj.2024.12.016","url":null,"abstract":"<p><p>The HIV-1 capsid is an irregularly shaped protein complex containing the viral genome and several proteins needed for integration into the host cell genome. Small molecules, such as the drug-like compound PF-3450074 (PF74) and the anionic sugar inositolhexakisphosphate (IP6), are known to impact capsid stability, although the mechanisms through which they do so remain unknown. In this study, we employed atomistic molecular dynamics simulations to study the impact of molecules bound to hexamers at the central pore (IP6) and the FG-binding site (PF74) on the interface between capsid oligomers. We found that the IP6 cofactor stabilizes a pair of neighboring hexamers in their flattest configurations, whereas PF74 introduces a strong preference for intermediate tilt angles. These results suggest that the tilt angle between neighboring hexamers is a primary mechanism for the modulation of capsid stability. In addition, hexamer-pentamer interfaces were highly stable, suggesting that pentamers are likely not the locus of disassembly.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142845756","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":"Probabilistic analysis of spatial viscoelastic cues in 3D cell culture using magnetic microrheometry.","authors":"Ossi Arasalo, Arttu J Lehtonen, Mari Kielosto, Markus Heinonen, Juho Pokki","doi":"10.1016/j.bpj.2024.12.010","DOIUrl":"10.1016/j.bpj.2024.12.010","url":null,"abstract":"<p><p>Breast tumors are typically surrounded by extracellular matrix (ECM), which is heterogeneous, not just structurally but also mechanically. Conventional rheometry is inadequate for describing cell-size-level spatial differences in ECM mechanics that are evident at micrometer scales. Optical tweezers and passive microrheometry provide a microscale resolution for the purpose but are incapable of measuring ECM viscoelasticity (the liquid-like viscous and solid-like elastic characteristics) at stiffness levels as found in breast tumor biopsies. Magnetic microrheometry records data on varying microscale viscoelasticity within 3D ECM-mimicking materials up to the biopsy-relevant stiffness. However, the measurement probe-based microrheometry data has limitations in spatial resolution. Here, we present a probabilistic modeling method-providing analysis of sparse, probe-based spatial information on microscale viscoelasticity in ECM obtained from magnetic microrheometry-in two parts. First, we validate the method's applicability for analysis of a controlled stiffness difference, based on two collagen type 1 concentrations in one sample, showing a detectable stiffness gradient in the interface of the changing concentrations. Second, we used the method to quantify and visualize differences in viscoelasticity within 3D cell cultures containing breast-cancer-associated fibroblasts, and collagen type 1 (both typically present in the tumor ECM). The fibroblasts' presence stiffens the collagen material, which aligns with previous research. Importantly, we provide probabilistic quantification of related spatial heterogeneity differences in viscoelasticity recorded by magnetic microrheometry, for the first time. The fibroblasts culturing leads to an initially higher spatial heterogeneity in the collagen stiffness. In summary, this method reports on enhanced spatial mapping of viscoelasticity in breast cancer 3D cultures, with the future potential for matching of spatial viscoelasticity distribution in the 3D cultures with the one in biopsies.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142845758","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}