首页 > 最新文献

bioRxiv - Biophysics最新文献

英文 中文
Stress-mediated growth determines E. coli division site morphogenesis 压力介导的生长决定了大肠杆菌分裂点的形态发生
Pub Date : 2024-09-12 DOI: 10.1101/2024.09.11.612282
Petr Pelech, Paula P Navarro, Andrea Vettiger, Luke H Chao, Christoph Allolio
In order to proliferate, bacteria must remodel their cell wall at the division site. The division process is driven by the enzymatic activity of peptidoglycan (PG) synthases and hydrolases around the constricting Z-ring. PG remodelling is regulated by de- and re-crosslinking enzymes, and the directing constrictive force of the Z ring. We introduce a model that is able to reproduce correctly the shape of the division site during the constriction and septation phase of E. coli. The model represents mechanochemical coupling within the mathematical framework of morphoelasticity. It contains only two adjustable parameters, associated with volumetric growth and PG remodelling, that are coupled to the mechanical stress in the bacterial wall. Different morphologies, corresponding either to mutant or wild type cells were recovered as a function of the remodeling parameter. In addition, a plausible range for the cell stiffness and turgor pressure was determined by comparing numerical simulations with bacterial cell plasmolysis data.
为了增殖,细菌必须在分裂部位重塑细胞壁。分裂过程由收缩 Z 环周围的肽聚糖(PG)合成酶和水解酶的酶活性驱动。肽聚糖的重塑受去交联和再交联酶以及 Z 环的定向收缩力的调节。我们介绍的模型能够正确再现大肠杆菌在收缩和隔膜阶段分裂部位的形状。该模型在形态弹性数学框架内体现了机械化学耦合。它只包含两个可调参数,分别与体积增长和 PG 重塑有关,并与细菌壁的机械应力相耦合。作为重塑参数的函数,恢复了突变型或野生型细胞的不同形态。此外,通过比较数值模拟和细菌细胞解痉数据,确定了细胞硬度和张力压力的合理范围。
{"title":"Stress-mediated growth determines E. coli division site morphogenesis","authors":"Petr Pelech, Paula P Navarro, Andrea Vettiger, Luke H Chao, Christoph Allolio","doi":"10.1101/2024.09.11.612282","DOIUrl":"https://doi.org/10.1101/2024.09.11.612282","url":null,"abstract":"In order to proliferate, bacteria must remodel their cell wall at the division site. The division process is driven by the enzymatic activity of peptidoglycan (PG) synthases and hydrolases around the constricting Z-ring. PG remodelling is regulated by de- and re-crosslinking enzymes, and the directing constrictive force of the Z ring. We introduce a model that is able to reproduce correctly the shape of the division site during the constriction and septation phase of <em>E. coli</em>. The model represents mechanochemical coupling within the mathematical framework of morphoelasticity. It contains only two adjustable parameters, associated with volumetric growth and PG remodelling, that are coupled to the mechanical stress in the bacterial wall. Different morphologies, corresponding either to mutant or wild type cells were recovered as a function of the remodeling parameter. In addition, a plausible range for the cell stiffness and turgor pressure was determined by comparing numerical simulations with bacterial cell plasmolysis data.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"133 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178169","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}
引用次数: 0
Myosin cluster dynamics determines epithelial wound ring constriction 肌球蛋白簇动力学决定上皮伤口环收缩
Pub Date : 2024-09-12 DOI: 10.1101/2024.09.12.612715
Alka Bhat, Remi Berthoz, Simon Lo Vecchio, Coralie Spiegelhalter, Shigenobu Yonemura, Olivier Pertz, Daniel Riveline
Collection of myosin motors and actin filaments can self-assemble into submicrometric clusters under the regulation of RhoA. Emergent dynamics of these clusters have been reported in a variety of morphogenetic systems, ranging from Drosophila to acto-myosin assays in vitro. In single cell cytokinetic rings, acto-myosin clusters are associated with stress generation when radial and transport when tangential with respect to the ring closure. Here, we show that these phenomena hold true for acto-myosin multi-cellular rings during wound closure in epithelial monolayers. We assessed the activity of RhoA using FRET sensors, and we report that cluster dynamics does not correlate with RhoA activity. Nevertheless, we show that bursts of RhoA activation precede recruitment of myosin. Altogether myosin clusters dynamics is conserved between single and multi-cellular systems and this suggests that they could be used as generic read-outs for mapping and predicting stress generation and shape changes in morphogenesis.
在 RhoA 的调控下,肌球蛋白马达和肌动蛋白丝的集合可以自组装成亚微米级的簇。从果蝇到体外肌动蛋白试验等各种形态发生系统都报道了这些集群的新动态。在单细胞细胞运动环中,肌动蛋白簇与环闭合时的径向应力产生和切向应力运输有关。在这里,我们发现在上皮单层细胞伤口闭合过程中,肌动蛋白多细胞环也存在这些现象。我们使用 FRET 传感器对 RhoA 的活性进行了评估,结果发现簇的动态与 RhoA 的活性并不相关。然而,我们发现在肌球蛋白招募之前会出现 RhoA 激活爆发。总之,肌球蛋白簇的动态在单细胞和多细胞系统之间是一致的,这表明它们可以用作绘制和预测形态发生过程中应力产生和形状变化的通用读出器。
{"title":"Myosin cluster dynamics determines epithelial wound ring constriction","authors":"Alka Bhat, Remi Berthoz, Simon Lo Vecchio, Coralie Spiegelhalter, Shigenobu Yonemura, Olivier Pertz, Daniel Riveline","doi":"10.1101/2024.09.12.612715","DOIUrl":"https://doi.org/10.1101/2024.09.12.612715","url":null,"abstract":"Collection of myosin motors and actin filaments can self-assemble into submicrometric clusters under the regulation of RhoA. Emergent dynamics of these clusters have been reported in a variety of morphogenetic systems, ranging from Drosophila to acto-myosin assays in vitro. In single cell cytokinetic rings, acto-myosin clusters are associated with stress generation when radial and transport when tangential with respect to the ring closure. Here, we show that these phenomena hold true for acto-myosin multi-cellular rings during wound closure in epithelial monolayers. We assessed the activity of RhoA using FRET sensors, and we report that cluster dynamics does not correlate with RhoA activity. Nevertheless, we show that bursts of RhoA activation precede recruitment of myosin. Altogether myosin clusters dynamics is conserved between single and multi-cellular systems and this suggests that they could be used as generic read-outs for mapping and predicting stress generation and shape changes in morphogenesis.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178155","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}
引用次数: 0
Allosteric modulation of proton binding confers Cl- activation and glutamate selectivity to vesicular glutamate transporters 质子结合的异构调节赋予囊泡谷氨酸转运体 Cl- 激活和谷氨酸选择性
Pub Date : 2024-09-12 DOI: 10.1101/2024.09.06.609381
Bart Borghans, Daniel Kortzak, Piersilvio Longo, Jan-Philipp Machtens, Christoph Fahlke
Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and remove luminal Cl- via an additional anion channel mode. Both of these transport functions are stimulated by luminal acidification, luminal-positive membrane potential, and luminal Cl-. We studied VGLUT1 transporter/channel activation using a combination of heterologous expression, cellular electrophysiology, fast solution exchange, and mathematical modeling. Cl- channel gating can be described with a kinetic scheme that includes two protonation sites and distinct opening, closing, and Cl--binding rates for each protonation state. Cl- binding promotes channel opening by modifying the pKa values of the protonation sites and rates of pore opening and closure. VGLUT1 transports glutamate and aspartate at distinct stoichiometries: H+-glutamate exchange at 1:1 stoichiometry and aspartate uniport. Neurotransmitter transport with variable stoichiometry can be described with an alternating access model that assumes that transporters without substrate translocate in the doubly protonated state to the inward-facing conformation and return with the bound amino acid substrate as either singly or doubly protonated. Glutamate, but not aspartate, promotes the release of one proton from inward-facing VGLUT1, resulting in preferential H+-coupled glutamate exchange. Cl- stimulates glutamate transport by making the glutamate-binding site accessible to cytoplasmic glutamate and by facilitating transitions to the inward-facing conformation after outward substrate release. We conclude that allosteric modification of transporter protonation by Cl- is crucial for both VGLUT1 transport functions.
囊泡谷氨酸转运体(VGLUTs)用谷氨酸填充突触囊泡,并通过另一种阴离子通道模式清除管腔内的 Cl-。这两种转运功能都受到管腔酸化、管腔正膜电位和管腔 Cl- 的刺激。我们采用异源表达、细胞电生理学、快速溶液交换和数学建模相结合的方法研究了 VGLUT1 转运体/通道活化。Cl- 通道门控可以用一个动力学方案来描述,该方案包括两个质子化位点以及每个质子化状态的不同打开、关闭和 Cl- 结合速率。Cl- 结合通过改变质子化位点的 pKa 值以及孔隙打开和关闭的速率来促进通道打开。VGLUT1 以不同的比例运输谷氨酸和天冬氨酸:以 1:1 的化学计量进行 H+-谷氨酸交换和天冬氨酸单端口。具有可变化学计量的神经递质转运可以用交替通路模型来描述,该模型假定不含底物的转运体以双质子化状态转运至内向构象,然后以单质子化或双质子化状态带着结合的氨基酸底物返回。谷氨酸(而非天门冬氨酸)可促进内向型 VGLUT1 释放一个质子,从而优先进行 H+耦合谷氨酸交换。Cl- 可使谷氨酸结合位点与胞浆谷氨酸接触,并在底物向外释放后促进向内构象的转换,从而刺激谷氨酸的转运。我们的结论是,Cl- 对转运体质子化的异构修饰对 VGLUT1 的两种转运功能都至关重要。
{"title":"Allosteric modulation of proton binding confers Cl- activation and glutamate selectivity to vesicular glutamate transporters","authors":"Bart Borghans, Daniel Kortzak, Piersilvio Longo, Jan-Philipp Machtens, Christoph Fahlke","doi":"10.1101/2024.09.06.609381","DOIUrl":"https://doi.org/10.1101/2024.09.06.609381","url":null,"abstract":"Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and remove luminal Cl<sup>-</sup> via an additional anion channel mode. Both of these transport functions are stimulated by luminal acidification, luminal-positive membrane potential, and luminal Cl<sup>-</sup>. We studied VGLUT1 transporter/channel activation using a combination of heterologous expression, cellular electrophysiology, fast solution exchange, and mathematical modeling. Cl<sup>-</sup> channel gating can be described with a kinetic scheme that includes two protonation sites and distinct opening, closing, and Cl<sup>-</sup>-binding rates for each protonation state. Cl<sup>-</sup> binding promotes channel opening by modifying the pKa values of the protonation sites and rates of pore opening and closure. VGLUT1 transports glutamate and aspartate at distinct stoichiometries: H<sup>+</sup>-glutamate exchange at 1:1 stoichiometry and aspartate uniport. Neurotransmitter transport with variable stoichiometry can be described with an alternating access model that assumes that transporters without substrate translocate in the doubly protonated state to the inward-facing conformation and return with the bound amino acid substrate as either singly or doubly protonated. Glutamate, but not aspartate, promotes the release of one proton from inward-facing VGLUT1, resulting in preferential H<sup>+</sup>-coupled glutamate exchange. Cl<sup>-</sup> stimulates glutamate transport by making the glutamate-binding site accessible to cytoplasmic glutamate and by facilitating transitions to the inward-facing conformation after outward substrate release. We conclude that allosteric modification of transporter protonation by Cl<sup>-</sup> is crucial for both VGLUT1 transport functions.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"108 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178170","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}
引用次数: 0
Symmetry of loop extrusion by dimeric SMC complexes is DNA-tension-dependent 二聚体 SMC 复合物挤出环的对称性取决于 DNA 张力
Pub Date : 2024-09-12 DOI: 10.1101/2024.09.12.612694
Biswajit Pradhan, Adrian Pinto, Takaharu Kanno, Damla Tetiker, Martin D. Baaske, Erin Cutt, Constantinos Chatzicharlampous, Herwig Schüler, Amar Deep, Kevin D. Corbett, Luis Aragon, Peter Virnau, Camilla Björkegren, Eugene Kim
Structural maintenance of chromosome (SMC) complexes organize and regulate genomes via DNA loop extrusion. During this process, the complexes increase the loop size by reeling in DNA from one or both sides of the loop. The factors governing this symmetry remain unclear. Here, we combine single-molecule analysis and molecular dynamic simulations to investigate the symmetry of loop extrusion of various SMC complexes. We find that whereas monomeric condensin and cohesin are one-sided extruders, the symmetry of dimeric SMCs, such as Smc5/6 and Wadjet, is DNA tension dependent. At low DNA tension (< 0.1pN), Smc5/6 and Wadjet extrude DNA from both sides of the loop. At higher tension, however, they transition to a behavior akin to one-sided extruders, yet still capable of extruding from one or the other side thereby switching the direction of extrusion. Our simulations further reveal that thermal fluctuations significantly influence loop extrusion symmetry, causing variations in DNA reeling rates between the two motors in the dimeric complexes and their direction switching at stalling tensions. Our findings challenge the previous view of loop extrusion symmetry as a fixed characteristic, revealing its dynamic nature and regulation by both intrinsic protein properties and extrinsic factors.
染色体结构维护(SMC)复合物通过 DNA 环挤压来组织和调节基因组。在这一过程中,复合体通过从环路的一侧或两侧卷入 DNA 来增加环路的大小。调节这种对称性的因素仍不清楚。在这里,我们结合单分子分析和分子动力学模拟来研究各种 SMC 复合物环挤压的对称性。我们发现,单体凝集素和凝聚素是单侧挤出的,而二聚体 SMC(如 Smc5/6 和 Wadjet)的对称性则取决于 DNA 张力。在 DNA 张力较低(0.1pN)时,Smc5/6 和 Wadjet 从环路两侧挤出 DNA。然而,在张力较高时,它们会过渡到类似于单侧挤出器的行为,但仍能从一侧或另一侧挤出,从而转换挤出方向。我们的模拟进一步揭示了热波动对环路挤压对称性的显著影响,导致二聚体复合物中两个马达之间的DNA卷绕率发生变化,以及它们在张力失速时的方向切换。我们的发现挑战了以往将环路挤压对称性视为固定特性的观点,揭示了其动态性质以及受蛋白质内在特性和外在因素的调控。
{"title":"Symmetry of loop extrusion by dimeric SMC complexes is DNA-tension-dependent","authors":"Biswajit Pradhan, Adrian Pinto, Takaharu Kanno, Damla Tetiker, Martin D. Baaske, Erin Cutt, Constantinos Chatzicharlampous, Herwig Schüler, Amar Deep, Kevin D. Corbett, Luis Aragon, Peter Virnau, Camilla Björkegren, Eugene Kim","doi":"10.1101/2024.09.12.612694","DOIUrl":"https://doi.org/10.1101/2024.09.12.612694","url":null,"abstract":"Structural maintenance of chromosome (SMC) complexes organize and regulate genomes via DNA loop extrusion. During this process, the complexes increase the loop size by reeling in DNA from one or both sides of the loop. The factors governing this symmetry remain unclear. Here, we combine single-molecule analysis and molecular dynamic simulations to investigate the symmetry of loop extrusion of various SMC complexes. We find that whereas monomeric condensin and cohesin are one-sided extruders, the symmetry of dimeric SMCs, such as Smc5/6 and Wadjet, is DNA tension dependent. At low DNA tension (&lt; 0.1pN), Smc5/6 and Wadjet extrude DNA from both sides of the loop. At higher tension, however, they transition to a behavior akin to one-sided extruders, yet still capable of extruding from one or the other side thereby switching the direction of extrusion. Our simulations further reveal that thermal fluctuations significantly influence loop extrusion symmetry, causing variations in DNA reeling rates between the two motors in the dimeric complexes and their direction switching at stalling tensions. Our findings challenge the previous view of loop extrusion symmetry as a fixed characteristic, revealing its dynamic nature and regulation by both intrinsic protein properties and extrinsic factors.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"25 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178168","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}
引用次数: 0
Molecular basis of the urate transporter URAT1 inhibition by gout drugs 痛风药物抑制尿酸盐转运体 URAT1 的分子基础
Pub Date : 2024-09-12 DOI: 10.1101/2024.09.11.612563
Yang Suo, Justin Fedor, Han Zhang, Kalina Tsolova, Xiaoyu Shi, Kedar Sharma, Shweta Kumari, Mario J. Borgnia, Peng Zhan, Wonpil Im, Seok-Yong Lee
Hyperuricemia is a condition when uric acid, a waste product of purine metabolism, accumulates in the blood. Untreated hyperuricemia can lead to crystal formation of monosodium urate in the joints, causing a painful inflammatory disease known as gout. These conditions are associated with many other diseases and affect a significant and increasing proportion of the population. The human urate transporter 1 (URAT1) is responsible for the reabsorption of ~90% of uric acid in the kidneys back into the blood, making it a primary target for treating hyperuricemia and gout. Despite decades of research and development, clinically available URAT1 inhibitors have limitations because the molecular basis of URAT1 inhibition by gout drugs remains unknown5. Here we present cryo-electron microscopy structures of URAT1 alone and in complex with three clinically relevant inhibitors: benzbromarone, lesinurad, and the novel compound TD-3. Together with functional experiments and molecular dynamics simulations, we reveal that these inhibitors bind selectively to URAT1 in inward-open states. Furthermore, we discover differences in the inhibitor dependent URAT1 conformations as well as interaction networks, which contribute to drug specificity. Our findings illuminate a general theme for URAT1 inhibition, paving the way for the design of next-generation URAT1 inhibitors in the treatment of gout and hyperuricemia.
高尿酸血症是指尿酸(一种嘌呤代谢的废物)在血液中积聚。高尿酸血症如不及时治疗,会在关节中形成单钠尿酸盐结晶,引起疼痛性炎症,即痛风。这些疾病与许多其他疾病相关,影响着越来越多的人。人尿酸盐转运体 1(URAT1)负责将肾脏中约 90% 的尿酸重吸收回血液,因此成为治疗高尿酸血症和痛风的主要靶点。尽管经过数十年的研究和开发,临床上可用的URAT1抑制剂仍有局限性,因为痛风药物抑制URAT1的分子基础仍然未知5。在此,我们展示了URAT1单独和与三种临床相关抑制剂(苯溴马隆、来西脲德和新型化合物TD-3)复合物的冷冻电子显微镜结构。通过功能实验和分子动力学模拟,我们发现这些抑制剂在内向开放状态下选择性地与 URAT1 结合。此外,我们还发现了抑制剂依赖的 URAT1 构象以及相互作用网络的差异,这有助于药物的特异性。我们的发现阐明了URAT1抑制的一般主题,为设计治疗痛风和高尿酸血症的下一代URAT1抑制剂铺平了道路。
{"title":"Molecular basis of the urate transporter URAT1 inhibition by gout drugs","authors":"Yang Suo, Justin Fedor, Han Zhang, Kalina Tsolova, Xiaoyu Shi, Kedar Sharma, Shweta Kumari, Mario J. Borgnia, Peng Zhan, Wonpil Im, Seok-Yong Lee","doi":"10.1101/2024.09.11.612563","DOIUrl":"https://doi.org/10.1101/2024.09.11.612563","url":null,"abstract":"Hyperuricemia is a condition when uric acid, a waste product of purine metabolism, accumulates in the blood. Untreated hyperuricemia can lead to crystal formation of monosodium urate in the joints, causing a painful inflammatory disease known as gout. These conditions are associated with many other diseases and affect a significant and increasing proportion of the population. The human urate transporter 1 (URAT1) is responsible for the reabsorption of ~90% of uric acid in the kidneys back into the blood, making it a primary target for treating hyperuricemia and gout. Despite decades of research and development, clinically available URAT1 inhibitors have limitations because the molecular basis of URAT1 inhibition by gout drugs remains unknown5. Here we present cryo-electron microscopy structures of URAT1 alone and in complex with three clinically relevant inhibitors: benzbromarone, lesinurad, and the novel compound TD-3. Together with functional experiments and molecular dynamics simulations, we reveal that these inhibitors bind selectively to URAT1 in inward-open states. Furthermore, we discover differences in the inhibitor dependent URAT1 conformations as well as interaction networks, which contribute to drug specificity. Our findings illuminate a general theme for URAT1 inhibition, paving the way for the design of next-generation URAT1 inhibitors in the treatment of gout and hyperuricemia.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178167","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}
引用次数: 0
Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1 神经元钙传感器Synaptotagmin-1对囊泡对接和融合孔的调控
Pub Date : 2024-09-12 DOI: 10.1101/2024.09.12.612660
Maria Tsemperouli, Sudheer Kumar Cheppali, Felix Rivera Molina, David Chetrit, Ane Landajuela, Derek Toomre, Erdem Karatekin
Synaptotagmin-1 (Syt1) is a major calcium sensor for rapid neurotransmitter release in neurons and hormone release in many neuroendocrine cells. It possesses two tandem cytosolic C2 domains that bind calcium, negatively charged phospholipids, and the neuronal SNARE complex. Calcium binding to Syt1 triggers exocytosis, but how this occurs is not well understood. Syt1 has additional roles in docking dense core vesicles (DCV) and synaptic vesicles (SV) to the plasma membrane (PM) and in regulating fusion pore dynamics. Thus, Syt1 perturbations could affect release through vesicle docking, fusion triggering, fusion pore regulation, or a combination of these. Here, using a human neuroendocrine cell line, we show that neutralization of highly conserved polybasic patches in either C2 domain of Syt1 impairs both DCV docking and efficient release of serotonin from DCVs. Interestingly, the same mutations resulted in larger fusion pores and faster release of serotonin during individual fusion events. Thus, Syt1's roles in vesicle docking, fusion triggering, and fusion pore control may be functionally related.
突触肽键-1(Syt1)是一种主要的钙传感器,可促进神经元中神经递质的快速释放和许多神经内分泌细胞中激素的释放。它具有两个串联的胞浆 C2 结构域,可结合钙、带负电荷的磷脂和神经元 SNARE 复合物。钙与 Syt1 结合会触发外泌,但这是如何发生的还不十分清楚。Syt1 在将致密核心囊泡 (DCV) 和突触囊泡 (SV) 与质膜 (PM) 接合以及调节融合孔动态方面也有其他作用。因此,Syt1 的扰动可能会通过囊泡对接、融合触发、融合孔调节或这些因素的组合来影响释放。在这里,我们利用一种人类神经内分泌细胞系证明,中和 Syt1 C2 结构域中的任何一个高度保守的多基点,都会影响 DCV 的对接和血清素从 DCV 中的有效释放。有趣的是,同样的突变导致了更大的融合孔和在单个融合事件中更快的血清素释放。因此,Syt1 在囊泡对接、融合触发和融合孔控制中的作用可能在功能上是相关的。
{"title":"Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1","authors":"Maria Tsemperouli, Sudheer Kumar Cheppali, Felix Rivera Molina, David Chetrit, Ane Landajuela, Derek Toomre, Erdem Karatekin","doi":"10.1101/2024.09.12.612660","DOIUrl":"https://doi.org/10.1101/2024.09.12.612660","url":null,"abstract":"Synaptotagmin-1 (Syt1) is a major calcium sensor for rapid neurotransmitter release in neurons and hormone release in many neuroendocrine cells. It possesses two tandem cytosolic C2 domains that bind calcium, negatively charged phospholipids, and the neuronal SNARE complex. Calcium binding to Syt1 triggers exocytosis, but how this occurs is not well understood. Syt1 has additional roles in docking dense core vesicles (DCV) and synaptic vesicles (SV) to the plasma membrane (PM) and in regulating fusion pore dynamics. Thus, Syt1 perturbations could affect release through vesicle docking, fusion triggering, fusion pore regulation, or a combination of these. Here, using a human neuroendocrine cell line, we show that neutralization of highly conserved polybasic patches in either C2 domain of Syt1 impairs both DCV docking and efficient release of serotonin from DCVs. Interestingly, the same mutations resulted in larger fusion pores and faster release of serotonin during individual fusion events. Thus, Syt1's roles in vesicle docking, fusion triggering, and fusion pore control may be functionally related.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178171","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}
引用次数: 0
Enzyme activation by urea reveals the interplay between conformational dynamics and substrate binding: a single-molecule FRET study 尿素激活酶揭示构象动力学与底物结合之间的相互作用:单分子 FRET 研究
Pub Date : 2024-09-11 DOI: 10.1101/2024.09.01.610662
David Scheerer, Dorit Levy, Remi Casier, Inbal Riven, Hisham Mazal, Gilad Haran
Proteins often harness extensive motions of domains and subunits to promote their function. Deciphering how these movements impact activity is key for understanding life's molecular machinery. The enzyme adenylate kinase is an intriguing example for this relationship; it ensures efficient catalysis by large-scale domain motions that lead to the enclosure of the bound substrates ATP and AMP. At high concentrations, AMP also operates as an allosteric inhibitor of the protein. Surprisingly, the enzyme is activated by urea, a compound commonly acting as a denaturant. Combining single-molecule FRET spectroscopy and enzymatic activity studies, we find that urea interferes with two key mechanisms that contribute to enzyme efficacy. First, urea promotes the open conformation of the enzyme, aiding the proper positioning of the substrates. Second, urea decreases AMP affinity, paradoxically facilitating a more efficient progression towards the catalytically active complex. These results signify the important interplay between conformational dynamics and chemical steps, including binding, in the activity of enzymes. State-of-the-art tools, such as single-molecule fluorescence spectroscopy, offer new insights into how enzymes balance different conformations to regulate activity.
蛋白质通常利用结构域和亚基的大量运动来促进其功能。破解这些运动如何影响活性是了解生命分子机制的关键。腺苷酸激酶就是这种关系的一个有趣例子;它通过大规模的结构域运动确保高效催化,从而导致结合底物 ATP 和 AMP 的封闭。在高浓度下,AMP 还能作为蛋白质的异构抑制剂发挥作用。令人惊讶的是,尿素这种通常用作变性剂的化合物也能激活这种酶。结合单分子 FRET 光谱和酶活性研究,我们发现尿素干扰了两种有助于提高酶功效的关键机制。首先,脲会促进酶的开放构象,帮助底物正确定位。其次,脲会降低 AMP 的亲和力,从而促进更有效地形成催化活性复合物。这些结果表明,在酶的活性中,构象动力学与化学步骤(包括结合)之间存在重要的相互作用。单分子荧光光谱学等最先进的工具为了解酶如何平衡不同构象以调节活性提供了新的视角。
{"title":"Enzyme activation by urea reveals the interplay between conformational dynamics and substrate binding: a single-molecule FRET study","authors":"David Scheerer, Dorit Levy, Remi Casier, Inbal Riven, Hisham Mazal, Gilad Haran","doi":"10.1101/2024.09.01.610662","DOIUrl":"https://doi.org/10.1101/2024.09.01.610662","url":null,"abstract":"Proteins often harness extensive motions of domains and subunits to promote their function. Deciphering how these movements impact activity is key for understanding life's molecular machinery. The enzyme adenylate kinase is an intriguing example for this relationship; it ensures efficient catalysis by large-scale domain motions that lead to the enclosure of the bound substrates ATP and AMP. At high concentrations, AMP also operates as an allosteric inhibitor of the protein. Surprisingly, the enzyme is activated by urea, a compound commonly acting as a denaturant. Combining single-molecule FRET spectroscopy and enzymatic activity studies, we find that urea interferes with two key mechanisms that contribute to enzyme efficacy. First, urea promotes the open conformation of the enzyme, aiding the proper positioning of the substrates. Second, urea decreases AMP affinity, paradoxically facilitating a more efficient progression towards the catalytically active complex. These results signify the important interplay between conformational dynamics and chemical steps, including binding, in the activity of enzymes. State-of-the-art tools, such as single-molecule fluorescence spectroscopy, offer new insights into how enzymes balance different conformations to regulate activity.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178173","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}
引用次数: 0
Structural characterization of an essential lipoprotein of Mycoplasma pneumoniae 肺炎支原体必需脂蛋白的结构特征
Pub Date : 2024-09-11 DOI: 10.1101/2024.09.05.611430
Irem Keles, Sina Manger, Mbuso Siyabonga Mantanya, Achilleas S Frangakis
Mycoplasma pneumoniae is a human pathogen causing atypical community-acquired pneumonia. It is a model for a minimal cell, known for its non-canonical use of surface proteins for host-cell adhesion through ectodomain shedding and antigenic variation of surface proteins to evade the host cell immune response. Mpn444 is an essential mycoplasma surface protein implicated in both processes. It is one of 46 lipoproteins of M. pneumoniae, none of which have been structurally or functionally characterized. Here, we report the structure of Mpn444 at 3.04 Å as well as the molecular architecture of the trimeric Mpn444 complex. Our experimental structure displays striking similarity to structure predictions of several other essential lipoproteins in M. pneumoniae and other related Mycoplasma species, suggesting it to have a specialized and conserved function. The essentiality and involvement of Mpn444 in host immune evasion makes our structure a target for the development of new treatment strategies against mycoplasma infections.
肺炎支原体是一种导致非典型社区获得性肺炎的人类病原体。它是一种最小细胞模型,因其非典型使用表面蛋白通过外膜脱落粘附宿主细胞和表面蛋白抗原变异逃避宿主细胞免疫反应而闻名。Mpn444 是一种重要的支原体表面蛋白,与这两个过程都有关系。它是肺炎霉菌的 46 种脂蛋白之一,但没有一种脂蛋白具有结构或功能特征。在此,我们报告了 Mpn444 的 3.04 Å 结构以及 Mpn444 三聚体复合物的分子结构。我们的实验结构与肺炎双球菌和其他相关支原体中其他几种必需脂蛋白的结构预测显示出惊人的相似性,这表明它具有专门和保守的功能。Mpn444 在宿主免疫逃避中的重要性和参与性使我们的结构成为开发新的支原体感染治疗策略的目标。
{"title":"Structural characterization of an essential lipoprotein of Mycoplasma pneumoniae","authors":"Irem Keles, Sina Manger, Mbuso Siyabonga Mantanya, Achilleas S Frangakis","doi":"10.1101/2024.09.05.611430","DOIUrl":"https://doi.org/10.1101/2024.09.05.611430","url":null,"abstract":"Mycoplasma pneumoniae is a human pathogen causing atypical community-acquired pneumonia. It is a model for a minimal cell, known for its non-canonical use of surface proteins for host-cell adhesion through ectodomain shedding and antigenic variation of surface proteins to evade the host cell immune response. Mpn444 is an essential mycoplasma surface protein implicated in both processes. It is one of 46 lipoproteins of M. pneumoniae, none of which have been structurally or functionally characterized. Here, we report the structure of Mpn444 at 3.04 Å as well as the molecular architecture of the trimeric Mpn444 complex. Our experimental structure displays striking similarity to structure predictions of several other essential lipoproteins in M. pneumoniae and other related Mycoplasma species, suggesting it to have a specialized and conserved function. The essentiality and involvement of Mpn444 in host immune evasion makes our structure a target for the development of new treatment strategies against mycoplasma infections.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"48 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178172","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}
引用次数: 0
NMR at operational temperature for resonance assignments of a PET degrading enzyme 工作温度下的 NMR,用于 PET 降解酶的共振分配
Pub Date : 2024-09-10 DOI: 10.1101/2024.09.10.612188
Valeria Gabrielli, Jelena Grga, Sabine Gavalda, Laura Perrot, Emmanuelle Boll, Guy Lippens, Cyril Charlier, Guy Lippens
PETases are enzymes that can break down the PET polymer in its constituent building blocks, and thereby recycle starting material for new high-quality plastics. NMR spectroscopy can help in the understanding and ultimately improvement of these PETases, but is always confronted with the lengthy step of acquisition and interpretation of triple resonance spectra for the spectral assignment. Here, we explore whether this step can be made more efficient by recording the spectra directly at high temperature, which also corresponds to more realistic working conditions for the enzyme. Taking the inactive variant of LCCICCG in which the Serine 165 has been replaced by an Alanine (LCCICCG-S165A) as an example, we evaluate spectral quality at 30C and 50C, and find that the latter condition greatly improves the Signal-to-Noise (S/N) ratio of the different spectra. As a result, we present an exhaustive backbone and side-chain assignment of LCCICCG-S165A based on a minimal set of triple resonance spectra acquired at 50C, that can act as a basis for future work on bio-structural studies on this PETase.
PET 酶是一种能将 PET 聚合物分解为其组成构件的酶,从而循环利用起始材料制造新的优质塑料。核磁共振光谱有助于了解并最终改进这些 PET 酶,但始终面临着获取和解释三重共振谱以进行光谱分配的漫长步骤。在此,我们探讨了是否可以通过在高温下直接记录光谱来提高这一步骤的效率,因为高温也符合酶的更实际工作条件。以丝氨酸 165 被丙氨酸取代的 LCCICCG 非活性变体(LCCICCG-S165A)为例,我们分别在 30C 和 50C 温度下对光谱质量进行了评估,结果发现后者大大提高了不同光谱的信噪比(S/N)。因此,我们根据在 50C 温度下获得的一组最小的三重共振谱,对 LCCICCG-S165A 进行了详尽的骨架和侧链分配,为今后对这种 PET 酶进行生物结构研究奠定了基础。
{"title":"NMR at operational temperature for resonance assignments of a PET degrading enzyme","authors":"Valeria Gabrielli, Jelena Grga, Sabine Gavalda, Laura Perrot, Emmanuelle Boll, Guy Lippens, Cyril Charlier, Guy Lippens","doi":"10.1101/2024.09.10.612188","DOIUrl":"https://doi.org/10.1101/2024.09.10.612188","url":null,"abstract":"PETases are enzymes that can break down the PET polymer in its constituent building blocks, and thereby recycle starting material for new high-quality plastics. NMR spectroscopy can help in the understanding and ultimately improvement of these PETases, but is always confronted with the lengthy step of acquisition and interpretation of triple resonance spectra for the spectral assignment. Here, we explore whether this step can be made more efficient by recording the spectra directly at high temperature, which also corresponds to more realistic working conditions for the enzyme. Taking the inactive variant of LCCICCG in which the Serine 165 has been replaced by an Alanine (LCCICCG-S165A) as an example, we evaluate spectral quality at 30C and 50C, and find that the latter condition greatly improves the Signal-to-Noise (S/N) ratio of the different spectra. As a result, we present an exhaustive backbone and side-chain assignment of LCCICCG-S165A based on a minimal set of triple resonance spectra acquired at 50C, that can act as a basis for future work on bio-structural studies on this PETase.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"61 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142223568","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}
引用次数: 0
Lessons learned during the journey of data: from experiment to model for predicting kinase affinity, selectivity, polypharmacology, and resistance 数据之旅中的经验教训:从实验到模型,预测激酶的亲和性、选择性、多药理学和抗药性
Pub Date : 2024-09-10 DOI: 10.1101/2024.09.10.612176
Raquel Lopez-Rios de Castro, Jaime Rodriguez-Guerra, David Schaller, Talia B. Kimber, Corey Taylor, Jessica B White, Michael Backenkohler, Alexander Payne, Ben Kaminow, Ivan Pulido, Sukrit Singh, Paula Linh Krammer, Guillermo Perez-Hernandez, Andrea Volkamer, John D. Chodera
Recent advances in machine learning (ML) are reshaping drug discovery. Structure-based ML methods use physically-inspired models to predict binding affinities from protein:ligand complexes. These methods promise to enable the integration of data for many related targets, which addresses issues related to data scarcity for single targets and could enable generalizable predictions for a broad range of targets, including mutants. In this work, we report our experiences in building KinoML, a novel framework for ML in target-based small molecule drug discovery with an emphasis on structure-enabled methods. KinoML focuses currently on kinases as the relative structural conservation of this protein superfamily, particularly in the kinase domain, means it is possible to leverage data from the entire superfamily to make structure-informed predictions about binding affinities, selectivities, and drug resistance. Some key lessons learned in building KinoML include: the importance of reproducible data collection and deposition, the harmonization of molecular data and featurization, and the choice of the right data format to ensure reusability and reproducibility of ML models. As a result, KinoML allows users to easily achieve three tasks: accessing and curating molecular data; featurizing this data with representations suitable for ML applications; and running reproducible ML experiments that require access to ligand, protein, and assay information to predict ligand affinity. Despite KinoML focusing on kinases, this framework can be applied to other proteins. The lessons reported here can help guide the development of platforms for structure-enabled ML in other areas of drug discovery.
机器学习(ML)的最新进展正在重塑药物发现。基于结构的 ML 方法使用物理启发模型来预测蛋白质配体复合物的结合亲和力。这些方法有望整合许多相关靶点的数据,从而解决与单一靶点数据稀缺有关的问题,并能对包括突变体在内的广泛靶点进行通用预测。在这项工作中,我们报告了我们在构建 KinoML 方面的经验,KinoML 是基于靶点的小分子药物发现中的 ML 新框架,重点是结构赋能方法。KinoML 目前的重点是激酶,因为这个蛋白质超家族的结构相对保守,尤其是在激酶结构域,这意味着有可能利用整个超家族的数据,对结合亲和力、选择性和耐药性进行结构性预测。在构建 KinoML 的过程中学到的一些关键经验包括:可重复数据收集和沉积的重要性、分子数据的统一和特征化,以及选择正确的数据格式以确保 ML 模型的可重用性和可重复性。因此,KinoML 能让用户轻松完成三项任务:访问和整理分子数据;用适合 ML 应用的表示方法对这些数据进行特征化;运行可重现的 ML 实验,这些实验需要访问配体、蛋白质和检测信息,以预测配体亲和力。尽管 KinoML 专注于激酶,但这一框架也可应用于其他蛋白质。这里报告的经验有助于指导其他药物发现领域的结构化 ML 平台的开发。
{"title":"Lessons learned during the journey of data: from experiment to model for predicting kinase affinity, selectivity, polypharmacology, and resistance","authors":"Raquel Lopez-Rios de Castro, Jaime Rodriguez-Guerra, David Schaller, Talia B. Kimber, Corey Taylor, Jessica B White, Michael Backenkohler, Alexander Payne, Ben Kaminow, Ivan Pulido, Sukrit Singh, Paula Linh Krammer, Guillermo Perez-Hernandez, Andrea Volkamer, John D. Chodera","doi":"10.1101/2024.09.10.612176","DOIUrl":"https://doi.org/10.1101/2024.09.10.612176","url":null,"abstract":"Recent advances in machine learning (ML) are reshaping drug discovery. Structure-based ML methods use physically-inspired models to predict binding affinities from protein:ligand complexes. These methods promise to enable the integration of data for many related targets, which addresses issues related to data scarcity for single targets and could enable generalizable predictions for a broad range of targets, including mutants. In this work, we report our experiences in building KinoML, a novel framework for ML in target-based small molecule drug discovery with an emphasis on structure-enabled methods. KinoML focuses currently on kinases as the relative structural conservation of this protein superfamily, particularly in the kinase domain, means it is possible to leverage data from the entire superfamily to make structure-informed predictions about binding affinities, selectivities, and drug resistance. Some key lessons learned in building KinoML include: the importance of reproducible data collection and deposition, the harmonization of molecular data and featurization, and the choice of the right data format to ensure reusability and reproducibility of ML models. As a result, KinoML allows users to easily achieve three tasks: accessing and curating molecular data; featurizing this data with representations suitable for ML applications; and running reproducible ML experiments that require access to ligand, protein, and assay information to predict ligand affinity. Despite KinoML focusing on kinases, this framework can be applied to other proteins. The lessons reported here can help guide the development of platforms for structure-enabled ML in other areas of drug discovery.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178175","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}
引用次数: 0
期刊
bioRxiv - Biophysics
全部 Acc. Chem. Res. ACS Applied Bio Materials ACS Appl. Electron. Mater. ACS Appl. Energy Mater. ACS Appl. Mater. Interfaces ACS Appl. Nano Mater. ACS Appl. Polym. Mater. ACS BIOMATER-SCI ENG ACS Catal. ACS Cent. Sci. ACS Chem. Biol. ACS Chemical Health & Safety ACS Chem. Neurosci. ACS Comb. Sci. ACS Earth Space Chem. ACS Energy Lett. ACS Infect. Dis. ACS Macro Lett. ACS Mater. Lett. ACS Med. Chem. Lett. ACS Nano ACS Omega ACS Photonics ACS Sens. ACS Sustainable Chem. Eng. ACS Synth. Biol. Anal. Chem. BIOCHEMISTRY-US Bioconjugate Chem. BIOMACROMOLECULES Chem. Res. Toxicol. Chem. Rev. Chem. Mater. CRYST GROWTH DES ENERG FUEL Environ. Sci. Technol. Environ. Sci. Technol. Lett. Eur. J. Inorg. Chem. IND ENG CHEM RES Inorg. Chem. J. Agric. Food. Chem. J. Chem. Eng. Data J. Chem. Educ. J. Chem. Inf. Model. J. Chem. Theory Comput. J. Med. Chem. J. Nat. Prod. J PROTEOME RES J. Am. Chem. Soc. LANGMUIR MACROMOLECULES Mol. Pharmaceutics Nano Lett. Org. Lett. ORG PROCESS RES DEV ORGANOMETALLICS J. Org. Chem. J. Phys. Chem. J. Phys. Chem. A J. Phys. Chem. B J. Phys. Chem. C J. Phys. Chem. Lett. Analyst Anal. Methods Biomater. Sci. Catal. Sci. Technol. Chem. Commun. Chem. Soc. Rev. CHEM EDUC RES PRACT CRYSTENGCOMM Dalton Trans. Energy Environ. Sci. ENVIRON SCI-NANO ENVIRON SCI-PROC IMP ENVIRON SCI-WAT RES Faraday Discuss. Food Funct. Green Chem. Inorg. Chem. Front. Integr. Biol. J. Anal. At. Spectrom. J. Mater. Chem. A J. Mater. Chem. B J. Mater. Chem. C Lab Chip Mater. Chem. Front. Mater. Horiz. MEDCHEMCOMM Metallomics Mol. Biosyst. Mol. Syst. Des. Eng. Nanoscale Nanoscale Horiz. Nat. Prod. Rep. New J. Chem. Org. Biomol. Chem. Org. Chem. Front. PHOTOCH PHOTOBIO SCI PCCP Polym. Chem.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1