Pub Date : 2024-10-01Epub Date: 2024-08-26DOI: 10.1016/j.bpj.2024.08.019
Heidi M J Weakly, Kent J Wilson, Gunnar J Goetz, Emily L Pruitt, Amy Li, Libin Xu, Sarah L Keller
Researchers choose different methods of making giant unilamellar vesicles to satisfy different constraints of their experimental designs. A challenge that arises when researchers use a variety of methods is that each method may produce vesicles with a different average lipid ratio, even if all experiments use lipids from a common stock mixture. Here, we use mass spectrometry to investigate ratios of lipids in vesicle solutions made by five common methods: electroformation on indium tin oxide slides, electroformation on platinum wires, gentle hydration, emulsion transfer, and extrusion. We made vesicles from either five-component or binary mixtures of lipids chosen to span a wide range of physical properties: di(18:1)PC, di(16:0)PC, di(18:1)PG, di(12:0)PE, and cholesterol. For a mixture of all five of these lipids, ITO electroformation, Pt electroformation, gentle hydration, and extrusion methods result in only minor shifts in lipid ratios (≤5 mol %) relative to a common stock solution. In contrast, emulsion transfer results in ∼80% less cholesterol than expected from the stock solution, which is counterbalanced by a surprising overabundance of saturated PC-lipid relative to all other phospholipids. Experiments using binary mixtures of saturated and unsaturated PC-lipids and cholesterol largely support results from the five-component mixture. In general, our results imply that experiments that increment lipid ratios in small steps will produce data that are highly sensitive to the technique used and to sample-to-sample variations. For example, sample-to-sample variations are ∼±2 mol % for five-component vesicles produced by a single technique. In contrast, experiments that explore larger increments in lipid ratio or that seek to explain general trends and new phenomena will be less sensitive to sample-to-sample variation and the method used.
研究人员选择不同的方法制造巨型单拉米尔囊泡,以满足其实验设计的不同限制。当研究人员使用多种方法时会遇到的一个挑战是,即使所有实验使用的脂质都来自共同的混合物,每种方法产生的囊泡的平均脂质比率也可能不同。在这里,我们使用质谱法研究了五种常用方法制成的囊泡溶液中的脂质比率:氧化铟锡载玻片电铸法、铂丝电铸法、温和水合法、乳液转移法和挤压法。我们用五组份或二元脂质混合物制作囊泡,这些脂质混合物的物理特性范围很广:二(18:1)PC、二(16:0)PC、二(18:1)PG、二(12:0)PE 和胆固醇。对于所有这五种脂质的混合物,ITO 电形成、铂电形成、温和水合和挤压方法只会导致脂质比例相对于普通储备溶液发生微小变化(≤ 5 摩尔%)。相比之下,乳液转移的结果是胆固醇含量比原液少 80%,而饱和 PC 脂相对于所有其他磷脂却出人意料地过多,从而抵消了胆固醇含量的减少。使用饱和和不饱和 PC 脂及胆固醇的二元混合物进行的实验在很大程度上支持 5 组分混合物的结果。总的来说,我们的结果表明,以小步递增脂质比率的实验所产生的数据对所使用的技术和样本间的变化高度敏感。例如,对于用单一技术生产的 5 组分囊泡,样本与样本之间的差异约为±2 摩尔%。相比之下,探索较大脂质比率增量或试图解释总体趋势和新现象的实验对样本间差异和所用方法的敏感度较低。
{"title":"Several common methods of making vesicles (except an emulsion method) capture intended lipid ratios.","authors":"Heidi M J Weakly, Kent J Wilson, Gunnar J Goetz, Emily L Pruitt, Amy Li, Libin Xu, Sarah L Keller","doi":"10.1016/j.bpj.2024.08.019","DOIUrl":"10.1016/j.bpj.2024.08.019","url":null,"abstract":"<p><p>Researchers choose different methods of making giant unilamellar vesicles to satisfy different constraints of their experimental designs. A challenge that arises when researchers use a variety of methods is that each method may produce vesicles with a different average lipid ratio, even if all experiments use lipids from a common stock mixture. Here, we use mass spectrometry to investigate ratios of lipids in vesicle solutions made by five common methods: electroformation on indium tin oxide slides, electroformation on platinum wires, gentle hydration, emulsion transfer, and extrusion. We made vesicles from either five-component or binary mixtures of lipids chosen to span a wide range of physical properties: di(18:1)PC, di(16:0)PC, di(18:1)PG, di(12:0)PE, and cholesterol. For a mixture of all five of these lipids, ITO electroformation, Pt electroformation, gentle hydration, and extrusion methods result in only minor shifts in lipid ratios (≤5 mol %) relative to a common stock solution. In contrast, emulsion transfer results in ∼80% less cholesterol than expected from the stock solution, which is counterbalanced by a surprising overabundance of saturated PC-lipid relative to all other phospholipids. Experiments using binary mixtures of saturated and unsaturated PC-lipids and cholesterol largely support results from the five-component mixture. In general, our results imply that experiments that increment lipid ratios in small steps will produce data that are highly sensitive to the technique used and to sample-to-sample variations. For example, sample-to-sample variations are ∼±2 mol % for five-component vesicles produced by a single technique. In contrast, experiments that explore larger increments in lipid ratio or that seek to explain general trends and new phenomena will be less sensitive to sample-to-sample variation and the method used.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480763/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079067","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01Epub Date: 2024-08-28DOI: 10.1016/j.bpj.2024.08.022
Luis Vollmers, Martin Zacharias
Protein-RNA interactions are crucially important for numerous cellular processes and often involve coupled folding and binding of peptide segments upon association. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes such a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics free energy simulations were used to calculate the absolute binding free energy of the N-peptide of bacteriophage P22 in complex with the boxB RNA hairpin motif at different salt concentrations and using two different water force field models. We obtained good agreement with experiment also at different salt concentrations for the TIP4P-D water model that has a stabilizing effect on unfolded protein structures. It allowed us to estimate the free energy contribution resulting from restricting the molecules' spatial and conformational freedom upon binding, which makes a large opposing contribution to binding. In a second set of umbrella sampling simulations to dissociate/associate the complex along a separation coordinate, we analyzed the onset of preorientation of the N-peptide and onset of structure formation relative to the RNA and its dependence on the salt concentration. Peptide orientation and conformational transitions are significantly coupled to the first contact formation between peptide and RNA. The initial contacts are mostly formed between peptide residues and the boxB hairpin loop nucleotides. A complete transition to an α-helical bound peptide conformation occurs only at a late stage of the binding process a few angstroms before the complexed state has been reached. However, the N-peptide orients also at distances beyond the contact distance such that the sizable positive charge points toward the RNA's center-of-mass. Our result may have important implications for understanding protein- and peptide-RNA complex formation frequently involving coupled folding and association processes.
蛋白质与 RNA 的相互作用对许多细胞过程都至关重要,而且往往涉及肽段在结合时的耦合折叠和结合。噬菌体的坚果利用位点(N)蛋白含有一个 N 端富含精氨酸的基团,在与 boxB RNA 发夹环目标结构结合时会发生这种折叠转变。我们利用分子动力学(MD)自由能模拟计算了噬菌体 P22 的 N 肽在不同盐浓度下与 boxB RNA 发夹图案复合时的绝对结合自由能,并使用了两种不同的水力场模型。在不同盐浓度条件下,我们发现 TIP4P-D 水模型与实验结果的一致性很好,该模型对未折叠蛋白质结构具有稳定作用。这使我们能够估算出在结合时限制分子的空间和构象自由度所产生的自由能贡献,这对结合有很大的反作用。在第二组伞状取样模拟中,我们沿分离坐标解离/分离了复合物,分析了 N 肽预取向的开始时间和相对于 RNA 的结构形成的开始时间及其对盐浓度的依赖性。肽的取向和构象转变与肽和 RNA 之间首次接触的形成密切相关。最初的接触主要是在多肽残基与 boxB 发夹环核苷酸之间形成的。只有在结合过程的晚期,即达到复合状态之前几埃处,才会完全转变为α螺旋结合肽构象。然而,N-肽也会在接触距离以外的地方定向,从而使相当大的正电荷指向 RNA 的质量中心。我们的研究结果可能对理解经常涉及耦合折叠和结合过程的蛋白质和肽-RNA复合物的形成具有重要意义。
{"title":"Advanced sampling simulations of coupled folding and binding of phage P22 N-peptide to boxB RNA.","authors":"Luis Vollmers, Martin Zacharias","doi":"10.1016/j.bpj.2024.08.022","DOIUrl":"10.1016/j.bpj.2024.08.022","url":null,"abstract":"<p><p>Protein-RNA interactions are crucially important for numerous cellular processes and often involve coupled folding and binding of peptide segments upon association. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes such a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics free energy simulations were used to calculate the absolute binding free energy of the N-peptide of bacteriophage P22 in complex with the boxB RNA hairpin motif at different salt concentrations and using two different water force field models. We obtained good agreement with experiment also at different salt concentrations for the TIP4P-D water model that has a stabilizing effect on unfolded protein structures. It allowed us to estimate the free energy contribution resulting from restricting the molecules' spatial and conformational freedom upon binding, which makes a large opposing contribution to binding. In a second set of umbrella sampling simulations to dissociate/associate the complex along a separation coordinate, we analyzed the onset of preorientation of the N-peptide and onset of structure formation relative to the RNA and its dependence on the salt concentration. Peptide orientation and conformational transitions are significantly coupled to the first contact formation between peptide and RNA. The initial contacts are mostly formed between peptide residues and the boxB hairpin loop nucleotides. A complete transition to an α-helical bound peptide conformation occurs only at a late stage of the binding process a few angstroms before the complexed state has been reached. However, the N-peptide orients also at distances beyond the contact distance such that the sizable positive charge points toward the RNA's center-of-mass. Our result may have important implications for understanding protein- and peptide-RNA complex formation frequently involving coupled folding and association processes.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480772/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142104003","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01Epub Date: 2024-08-19DOI: 10.1016/j.bpj.2024.08.009
Zenghao Zhu, Wisam Reid, Shefin Sam George, Victoria Ou, Dáibhid Ó Maoiléidigh
In mammals, outer-hair-cell hair bundles (OHBs) transduce sound-induced forces into receptor currents and are required for the wide dynamic range and high sensitivity of hearing. OHBs differ conspicuously in morphology from other types of bundles. Here, we show that the 3D morphology of an OHB greatly impacts its mechanics and transduction. An OHB comprises rod-like stereocilia, which pivot on the surface of its sensory outer hair cell. Stereocilium pivot positions are arranged in columns and form a V shape. We measure the pivot positions and determine that OHB columns are far from parallel. To calculate the consequences of an OHB's V shape and far-from-parallel columns, we develop a mathematical model of an OHB that relates its pivot positions, 3D morphology, mechanics, and receptor current. We find that the 3D morphology of the OHB can halve its stiffness, can double its damping coefficient, and causes stereocilium displacements driven by stimulus forces to differ substantially across the OHB. Stereocilium displacements drive the opening and closing of ion channels through which the receptor current flows. Owing to the stereocilium-displacement differences, the currents passing through the ion channels can peak versus the stimulus frequency and vary considerably across the OHB. Consequently, the receptor current peaks versus the stimulus frequency. Ultimately, the OHB's 3D morphology can increase its receptor-current dynamic range more than twofold. Our findings imply that potential pivot-position changes owing to development, mutations, or location within the mammalian auditory organ might greatly alter OHB function.
{"title":"3D morphology of an outer-hair-cell hair bundle increases its displacement and dynamic range.","authors":"Zenghao Zhu, Wisam Reid, Shefin Sam George, Victoria Ou, Dáibhid Ó Maoiléidigh","doi":"10.1016/j.bpj.2024.08.009","DOIUrl":"10.1016/j.bpj.2024.08.009","url":null,"abstract":"<p><p>In mammals, outer-hair-cell hair bundles (OHBs) transduce sound-induced forces into receptor currents and are required for the wide dynamic range and high sensitivity of hearing. OHBs differ conspicuously in morphology from other types of bundles. Here, we show that the 3D morphology of an OHB greatly impacts its mechanics and transduction. An OHB comprises rod-like stereocilia, which pivot on the surface of its sensory outer hair cell. Stereocilium pivot positions are arranged in columns and form a V shape. We measure the pivot positions and determine that OHB columns are far from parallel. To calculate the consequences of an OHB's V shape and far-from-parallel columns, we develop a mathematical model of an OHB that relates its pivot positions, 3D morphology, mechanics, and receptor current. We find that the 3D morphology of the OHB can halve its stiffness, can double its damping coefficient, and causes stereocilium displacements driven by stimulus forces to differ substantially across the OHB. Stereocilium displacements drive the opening and closing of ion channels through which the receptor current flows. Owing to the stereocilium-displacement differences, the currents passing through the ion channels can peak versus the stimulus frequency and vary considerably across the OHB. Consequently, the receptor current peaks versus the stimulus frequency. Ultimately, the OHB's 3D morphology can increase its receptor-current dynamic range more than twofold. Our findings imply that potential pivot-position changes owing to development, mutations, or location within the mammalian auditory organ might greatly alter OHB function.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480765/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142003562","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01Epub Date: 2024-08-05DOI: 10.1016/j.bpj.2024.07.042
Aurelia Bucciarelli, Alberto Mantegazza, Andreas Haeberlin, Dominik Obrist
Despite increased interest in the effect of lingering red blood cells (LRBCs) on the heterogeneous hematocrit distribution in the microcirculation, quantitative data on LRBCs before and after the lingering event are still limited. The aim of the study was to investigate the relation between red blood cell (RBC) lingering and hematocrit partitioning in a microfluidic model of a microvascular bifurcation in the limit of low hematocrit conditions (tube hematocrit <10%). To this end, the classification of LRBCs was performed based on timing, position, and velocity of the RBCs. The investigation provided statistical information on the velocity, shape, and orientation of LRBCs as well as on their lateral distribution in the parent and daughter vessels. LRBCs traveled predominantly close to the centerline of the parent vessel, but they marginated close to the distal wall in the daughter vessels. Differently than the RBC flow observed in the smallest vessels, no influence of lingering events on the local hematocrit partitioning was observed in our experiments. However, importantly, we found that LRBCs flowing in the daughter vessel after lingering may be connected to reverse hematocrit partitioning in downstream bifurcations by influencing the skewness of the hematocrit distribution in the daughter vessel, which relates to the so-called network history effect.
{"title":"Relation between hematocrit partitioning and red blood cell lingering in a microfluidic network.","authors":"Aurelia Bucciarelli, Alberto Mantegazza, Andreas Haeberlin, Dominik Obrist","doi":"10.1016/j.bpj.2024.07.042","DOIUrl":"10.1016/j.bpj.2024.07.042","url":null,"abstract":"<p><p>Despite increased interest in the effect of lingering red blood cells (LRBCs) on the heterogeneous hematocrit distribution in the microcirculation, quantitative data on LRBCs before and after the lingering event are still limited. The aim of the study was to investigate the relation between red blood cell (RBC) lingering and hematocrit partitioning in a microfluidic model of a microvascular bifurcation in the limit of low hematocrit conditions (tube hematocrit <10%). To this end, the classification of LRBCs was performed based on timing, position, and velocity of the RBCs. The investigation provided statistical information on the velocity, shape, and orientation of LRBCs as well as on their lateral distribution in the parent and daughter vessels. LRBCs traveled predominantly close to the centerline of the parent vessel, but they marginated close to the distal wall in the daughter vessels. Differently than the RBC flow observed in the smallest vessels, no influence of lingering events on the local hematocrit partitioning was observed in our experiments. However, importantly, we found that LRBCs flowing in the daughter vessel after lingering may be connected to reverse hematocrit partitioning in downstream bifurcations by influencing the skewness of the hematocrit distribution in the daughter vessel, which relates to the so-called network history effect.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480766/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141892801","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01Epub Date: 2024-08-14DOI: 10.1016/j.bpj.2024.08.007
Wen Cai, Karl Grosh
The outer hair cells (OHCs) of the mammalian cochlea are the mediators of an active, nonlinear electromechanical process necessary for sensitive, frequency-specific hearing. The membrane protein prestin conveys to the OHC a piezoelectric-like behavior hypothesized to actuate a high frequency, cycle-by-cycle conversion of electrical to mechanical energy to boost cochlear responses to low-level sound. This hypothesis has been debated for decades, with two key remaining issues: the influence of the rate dependence of conformal changes in prestin and the OHC transmembrane impedance. In this paper, we mainly focus on the rate dependence of the conformal change in prestin. A theoretical electromechanical model of the OHC that explicitly includes rate dependence of conformal transitions, viscoelasticity, and piezoelectricity. Using this theory, we show the influence of rate dependence and viscoelasticity on electromechanical force generation and transmembrane impedance. Furthermore, we stress the importance of using the correct mechanical boundary conditions when estimating the transmembrane capacitance. Finally, a set of experiments is described to uniquely estimate the constitutive properties of the OHC from whole-cell measurements.
{"title":"Rate-dependent cochlear outer hair cell force generation: Models and parameter estimation.","authors":"Wen Cai, Karl Grosh","doi":"10.1016/j.bpj.2024.08.007","DOIUrl":"10.1016/j.bpj.2024.08.007","url":null,"abstract":"<p><p>The outer hair cells (OHCs) of the mammalian cochlea are the mediators of an active, nonlinear electromechanical process necessary for sensitive, frequency-specific hearing. The membrane protein prestin conveys to the OHC a piezoelectric-like behavior hypothesized to actuate a high frequency, cycle-by-cycle conversion of electrical to mechanical energy to boost cochlear responses to low-level sound. This hypothesis has been debated for decades, with two key remaining issues: the influence of the rate dependence of conformal changes in prestin and the OHC transmembrane impedance. In this paper, we mainly focus on the rate dependence of the conformal change in prestin. A theoretical electromechanical model of the OHC that explicitly includes rate dependence of conformal transitions, viscoelasticity, and piezoelectricity. Using this theory, we show the influence of rate dependence and viscoelasticity on electromechanical force generation and transmembrane impedance. Furthermore, we stress the importance of using the correct mechanical boundary conditions when estimating the transmembrane capacitance. Finally, a set of experiments is described to uniquely estimate the constitutive properties of the OHC from whole-cell measurements.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480764/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141987361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01Epub Date: 2024-06-17DOI: 10.1016/j.bpj.2024.06.014
Vilmos Zsolnay, Margaret L Gardel, David R Kovar, Gregory A Voth
Actin filament networks are exposed to mechanical stimuli, but the effect of strain on actin filament structure has not been well established in molecular detail. This is a critical gap in understanding because the activity of a variety of actin-binding proteins has recently been determined to be altered by actin filament strain. We therefore used all-atom molecular dynamics simulations to apply tensile strains to actin filaments and find that changes in actin subunit organization are minimal in mechanically strained, but intact, actin filaments. However, a conformational change disrupts the critical D-loop to W-loop connection between longitudinal neighboring subunits, which leads to a metastable cracked conformation of the actin filament whereby one protofilament is broken prior to filament severing. We propose that the metastable crack presents a force-activated binding site for actin regulatory factors that specifically associate with strained actin filaments. Through protein-protein docking simulations, we find that 43 evolutionarily diverse members of the dual zinc-finger-containing LIM-domain family, which localize to mechanically strained actin filaments, recognize two binding sites exposed at the cracked interface. Furthermore, through its interactions with the crack, LIM domains increase the length of time damaged filaments remain stable. Our findings propose a new molecular model for mechanosensitive binding to actin filaments.
肌动蛋白丝网络暴露于机械刺激之下,但应变对肌动蛋白丝结构的影响尚未在分子细节方面得到很好的证实。这是一个重要的认识空白,因为最近已确定多种肌动蛋白结合蛋白的活性会因肌动蛋白丝应变而改变。因此,我们使用全原子分子动力学模拟对肌动蛋白丝施加拉伸应变,发现在机械应变但完好无损的肌动蛋白丝中,肌动蛋白亚基组织的变化微乎其微。然而,构象变化会破坏纵向相邻亚基之间关键的 D 环至 W 环连接,从而导致肌动蛋白丝出现可移动的裂缝构象,即在丝切断之前有一条原丝断裂。我们提出,可移动裂缝是肌动蛋白调控因子受力激活的结合位点,这些因子会与受拉伸的肌动蛋白丝发生特异性结合。通过蛋白质-蛋白质对接模拟,我们发现含有 LIM 结构域的双锌指家族的 43 个进化多样的成员会定位到机械应变的肌动蛋白丝上,它们能识别暴露在裂纹界面上的两个结合位点。此外,通过与裂纹的相互作用,LIM 结构域能延长受损肌动蛋白丝保持稳定的时间。我们的发现为肌动蛋白丝的机械敏感结合提出了一个新的分子模型。
{"title":"Cracked actin filaments as mechanosensitive receptors.","authors":"Vilmos Zsolnay, Margaret L Gardel, David R Kovar, Gregory A Voth","doi":"10.1016/j.bpj.2024.06.014","DOIUrl":"10.1016/j.bpj.2024.06.014","url":null,"abstract":"<p><p>Actin filament networks are exposed to mechanical stimuli, but the effect of strain on actin filament structure has not been well established in molecular detail. This is a critical gap in understanding because the activity of a variety of actin-binding proteins has recently been determined to be altered by actin filament strain. We therefore used all-atom molecular dynamics simulations to apply tensile strains to actin filaments and find that changes in actin subunit organization are minimal in mechanically strained, but intact, actin filaments. However, a conformational change disrupts the critical D-loop to W-loop connection between longitudinal neighboring subunits, which leads to a metastable cracked conformation of the actin filament whereby one protofilament is broken prior to filament severing. We propose that the metastable crack presents a force-activated binding site for actin regulatory factors that specifically associate with strained actin filaments. Through protein-protein docking simulations, we find that 43 evolutionarily diverse members of the dual zinc-finger-containing LIM-domain family, which localize to mechanically strained actin filaments, recognize two binding sites exposed at the cracked interface. Furthermore, through its interactions with the crack, LIM domains increase the length of time damaged filaments remain stable. Our findings propose a new molecular model for mechanosensitive binding to actin filaments.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480757/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141417553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01Epub Date: 2024-08-08DOI: 10.1016/j.bpj.2024.08.003
Marco De Corato, Maria Jose Gomez-Benito
The nucleus of eukaryotic cells is constantly subjected to different kinds of mechanical stimuli, which can impact the organization of chromatin and, subsequently, the expression of genetic information. Experiments from different groups showed that nuclear deformation can lead to transient or permanent condensation or decondensation of chromatin and the mechanical activation of genes, thus altering the transcription of proteins. Changes in chromatin organization, in turn, change the mechanical properties of the nucleus, possibly leading to an auxetic behavior. Here, we model the mechanics of the nucleus as a chemically active polymer gel in which the chromatin can exist in two states: a self-attractive state representing the heterochromatin and a repulsive state representing euchromatin. The model predicts reversible or irreversible changes in chromatin condensation levels upon external deformations of the nucleus. We find an auxetic response for a broad range of parameters under small and large deformations. These results agree with experimental observations and highlight the key role of chromatin organization in the mechanical response of the nucleus.
{"title":"Interplay of chromatin organization and mechanics of the cell nucleus.","authors":"Marco De Corato, Maria Jose Gomez-Benito","doi":"10.1016/j.bpj.2024.08.003","DOIUrl":"10.1016/j.bpj.2024.08.003","url":null,"abstract":"<p><p>The nucleus of eukaryotic cells is constantly subjected to different kinds of mechanical stimuli, which can impact the organization of chromatin and, subsequently, the expression of genetic information. Experiments from different groups showed that nuclear deformation can lead to transient or permanent condensation or decondensation of chromatin and the mechanical activation of genes, thus altering the transcription of proteins. Changes in chromatin organization, in turn, change the mechanical properties of the nucleus, possibly leading to an auxetic behavior. Here, we model the mechanics of the nucleus as a chemically active polymer gel in which the chromatin can exist in two states: a self-attractive state representing the heterochromatin and a repulsive state representing euchromatin. The model predicts reversible or irreversible changes in chromatin condensation levels upon external deformations of the nucleus. We find an auxetic response for a broad range of parameters under small and large deformations. These results agree with experimental observations and highlight the key role of chromatin organization in the mechanical response of the nucleus.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480768/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141911576","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The surface of a cell is crowded with membrane proteins. The size, shape, density, and mobility of extracellular surface proteins mediate cell surface accessibility to external molecules, viral particles, and other cells. However, predicting these qualities is not always straightforward, even when protein structures are known. We previously developed an experimental method for measuring flow-driven lateral transport of neutravidin bound to biotinylated lipids in supported lipid bilayers. Here, we use this method to detect hydrodynamic force applied to a series of lipid-anchored proteins with increasing size. We find that the measured force reflects both protein size and shape, making it possible to distinguish these features of intact, folded proteins in their undisturbed orientation and proximity to the lipid membrane. In addition, our results demonstrate that individual proteins are transported large distances by flow forces on the order of femtoNewtons, similar in magnitude to the shear forces resulting from blood circulation or from the swimming motion of microorganisms. Similar protein transport across living cells by hydrodynamic force may contribute to biological flow sensing.
{"title":"Microfluidic measurement of the size and shape of lipid-anchored proteins.","authors":"Sreeja Sasidharan, Leah Knepper, Emily Ankrom, Gabriel Cucé, Lingyang Kong, Amanda Ratajczak, Wonpil Im, Damien Thévenin, Aurelia Honerkamp-Smith","doi":"10.1016/j.bpj.2024.08.026","DOIUrl":"10.1016/j.bpj.2024.08.026","url":null,"abstract":"<p><p>The surface of a cell is crowded with membrane proteins. The size, shape, density, and mobility of extracellular surface proteins mediate cell surface accessibility to external molecules, viral particles, and other cells. However, predicting these qualities is not always straightforward, even when protein structures are known. We previously developed an experimental method for measuring flow-driven lateral transport of neutravidin bound to biotinylated lipids in supported lipid bilayers. Here, we use this method to detect hydrodynamic force applied to a series of lipid-anchored proteins with increasing size. We find that the measured force reflects both protein size and shape, making it possible to distinguish these features of intact, folded proteins in their undisturbed orientation and proximity to the lipid membrane. In addition, our results demonstrate that individual proteins are transported large distances by flow forces on the order of femtoNewtons, similar in magnitude to the shear forces resulting from blood circulation or from the swimming motion of microorganisms. Similar protein transport across living cells by hydrodynamic force may contribute to biological flow sensing.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480770/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142124722","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01Epub Date: 2024-08-19DOI: 10.1016/j.bpj.2024.08.006
Edward Mendez-Otalvaro, Wojciech Kopec, Bert L de Groot
TWIK-related potassium channel 1 (TREK1), a two-pore-domain mammalian potassium (K+) channel, regulates the resting potential across cell membranes, presenting a promising therapeutic target for neuropathy treatment. The gating of this channel converges in the conformation of the narrowest part of the pore: the selectivity filter (SF). Various hypotheses explain TREK1 gating modulation, including the dynamics of loops connecting the SF with transmembrane helices and the stability of hydrogen bond (HB) networks adjacent to the SF. Recently, two small molecules (Q6F and Q5F) were reported as activators that affect TREK1 by increasing its open probability in single-channel current measurements. Here, using molecular dynamics simulations, we investigate the effect of these ligands on the previously proposed modulation mechanisms of TREK1 gating compared to the apo channel. Our findings reveal that loop dynamics at the upper region of the SF exhibit only a weak correlation with permeation events/nonpermeation periods, whereas the HB network behind the SF appears more correlated. These nonpermeation periods arise from both distinct mechanisms: a C-type inactivation (resulting from dilation at the top of the SF), which has been described previously, and a carbonyl flipping in an SF binding site. We find that, besides the prevention of C-type inactivation in the channel, the ligands increase the probability of permeation by modulating the dynamics of the carbonyl flipping, influenced by a threonine residue at the bottom of the SF. These results offer insights for rational ligand design to optimize the gating modulation of TREK1 and related K+ channels.
TREK1是一种双孔域(2P)哺乳动物钾(K+)通道,可调节细胞膜上的静息电位,是治疗神经病变的一个很有前景的靶点。该通道的门控收敛于孔道最窄部分的构象:选择性滤波器(SF)。有多种假说可以解释 TREK1 的门控调节,包括连接 SF 与跨膜螺旋的环的动力学以及邻近 SF 的氢键(HB)网络的稳定性。最近,有报道称两种小分子(Q6F和Q5F)可作为激活剂,在单通道电流测量中通过增加其开放概率来影响TREK1。在此,我们利用分子动力学(MD)模拟研究了这些配体对之前提出的 TREK1 门控机制的影响。我们的研究结果表明,SF 上部区域的环路动力学与渗透事件/非渗透期仅表现出微弱的相关性,而 SF 后面的 HB 网络则表现出更强的相关性。这些非渗透期来自两种不同的机制:一种是 C 型失活(由 SF 顶部的扩张引起),这在之前已有描述;另一种是 SF 结合位点的羰基翻转。我们发现,除了防止通道中的 C 型失活外,配体还能通过调节羰基翻转的动态来提高渗透概率,而羰基翻转则受到 SF 底部一个苏氨酸残基的影响。这些结果为合理设计配体以优化 TREK1 和相关 K+ 通道的门控调节提供了启示。
{"title":"Effect of two activators on the gating of a K<sub>2P</sub> channel.","authors":"Edward Mendez-Otalvaro, Wojciech Kopec, Bert L de Groot","doi":"10.1016/j.bpj.2024.08.006","DOIUrl":"10.1016/j.bpj.2024.08.006","url":null,"abstract":"<p><p>TWIK-related potassium channel 1 (TREK1), a two-pore-domain mammalian potassium (K<sup>+</sup>) channel, regulates the resting potential across cell membranes, presenting a promising therapeutic target for neuropathy treatment. The gating of this channel converges in the conformation of the narrowest part of the pore: the selectivity filter (SF). Various hypotheses explain TREK1 gating modulation, including the dynamics of loops connecting the SF with transmembrane helices and the stability of hydrogen bond (HB) networks adjacent to the SF. Recently, two small molecules (Q6F and Q5F) were reported as activators that affect TREK1 by increasing its open probability in single-channel current measurements. Here, using molecular dynamics simulations, we investigate the effect of these ligands on the previously proposed modulation mechanisms of TREK1 gating compared to the apo channel. Our findings reveal that loop dynamics at the upper region of the SF exhibit only a weak correlation with permeation events/nonpermeation periods, whereas the HB network behind the SF appears more correlated. These nonpermeation periods arise from both distinct mechanisms: a C-type inactivation (resulting from dilation at the top of the SF), which has been described previously, and a carbonyl flipping in an SF binding site. We find that, besides the prevention of C-type inactivation in the channel, the ligands increase the probability of permeation by modulating the dynamics of the carbonyl flipping, influenced by a threonine residue at the bottom of the SF. These results offer insights for rational ligand design to optimize the gating modulation of TREK1 and related K<sup>+</sup> channels.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480771/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142003563","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01Epub Date: 2024-07-30DOI: 10.1016/j.bpj.2024.07.035
Callum M Ives, Alp Tegin Şahin, Neil J Thomson, Ulrich Zachariae
A key capability of ion channels is the facilitation of selective permeation of certain ionic species across cellular membranes at high rates. Due to their physiological significance, ion channels are of great pharmaceutical interest as drug targets. The polymodal signal-detecting transient receptor potential (TRP) superfamily of ion channels forms a particularly promising group of drug targets. While most members of this family permeate a broad range of cations including Ca2+, TRPM4 and TRPM5 are unique due to their strong monovalent selectivity and impermeability for divalent cations. Here, we investigated the mechanistic basis for their unique monovalent selectivity by in silico electrophysiology simulations of TRPM5. Our simulations reveal an unusual mechanism of cation selectivity, which is underpinned by the function of the central channel cavity alongside the selectivity filter. Our results suggest that a subtle hydrophobic barrier at the cavity entrance ("hydrophobic funnel") enables monovalent but not divalent cations to pass and occupy the cavity at physiologically relevant membrane voltages. Monovalent cations then permeate efficiently by a cooperative, distant knock-on mechanism between two binding regions in the extracellular pore vestibule and the central cavity. By contrast, divalent cations do not enter or interact favorably with the channel cavity due to its raised hydrophobicity. Hydrophilic mutations in the transition zone between the selectivity filter and the central channel cavity abolish the barrier for divalent cations, enabling both monovalent and divalent cations to traverse TRPM5.
{"title":"A hydrophobic funnel governs monovalent cation selectivity in the ion channel TRPM5.","authors":"Callum M Ives, Alp Tegin Şahin, Neil J Thomson, Ulrich Zachariae","doi":"10.1016/j.bpj.2024.07.035","DOIUrl":"10.1016/j.bpj.2024.07.035","url":null,"abstract":"<p><p>A key capability of ion channels is the facilitation of selective permeation of certain ionic species across cellular membranes at high rates. Due to their physiological significance, ion channels are of great pharmaceutical interest as drug targets. The polymodal signal-detecting transient receptor potential (TRP) superfamily of ion channels forms a particularly promising group of drug targets. While most members of this family permeate a broad range of cations including Ca<sup>2+</sup>, TRPM4 and TRPM5 are unique due to their strong monovalent selectivity and impermeability for divalent cations. Here, we investigated the mechanistic basis for their unique monovalent selectivity by in silico electrophysiology simulations of TRPM5. Our simulations reveal an unusual mechanism of cation selectivity, which is underpinned by the function of the central channel cavity alongside the selectivity filter. Our results suggest that a subtle hydrophobic barrier at the cavity entrance (\"hydrophobic funnel\") enables monovalent but not divalent cations to pass and occupy the cavity at physiologically relevant membrane voltages. Monovalent cations then permeate efficiently by a cooperative, distant knock-on mechanism between two binding regions in the extracellular pore vestibule and the central cavity. By contrast, divalent cations do not enter or interact favorably with the channel cavity due to its raised hydrophobicity. Hydrophilic mutations in the transition zone between the selectivity filter and the central channel cavity abolish the barrier for divalent cations, enabling both monovalent and divalent cations to traverse TRPM5.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480762/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141858922","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}