Pub Date : 2024-09-10DOI: 10.1101/2024.09.10.612206
Gerhard Michael Artmann, Oliver H. Weiergraeber, Samar Abdullah M. Damiati, Ipek Seda Firat, Aysegul Artmann
We propose the Interfacial Water Quantum-transition model (IWQ model) explaining temperature-dependent functional transitions in proteins. The model postulates that measured critical temperatures, TC, correspond to reference temperatures, TW, defined by rotational quantum transitions of temporarily free water molecules at the protein-water interface. The model's applicability is demonstrated through transitions in hemoglobin and thermosensitive TRP channels. We suggest this mechanism also defines basal body temperatures in homeotherms, with TW=36.32 degrees C for humans. We demonstrate that human (mammal) and chicken (Aves) body temperatures align with specific reference temperatures, and correlate with pronounced transitions at TC in hemoglobin oxygen saturation. This suggests evolutionary adaptations in homeotherms involve an interplay between oxygen supply and water's rotational transition temperatures. The IWQ-model states that proteins sense and water sets critical physiological temperatures.
{"title":"The molecular origin of body temperature in homeothermic species","authors":"Gerhard Michael Artmann, Oliver H. Weiergraeber, Samar Abdullah M. Damiati, Ipek Seda Firat, Aysegul Artmann","doi":"10.1101/2024.09.10.612206","DOIUrl":"https://doi.org/10.1101/2024.09.10.612206","url":null,"abstract":"We propose the Interfacial Water Quantum-transition model (IWQ model) explaining temperature-dependent functional transitions in proteins. The model postulates that measured critical temperatures, TC, correspond to reference temperatures, TW, defined by rotational quantum transitions of temporarily free water molecules at the protein-water interface. The model's applicability is demonstrated through transitions in hemoglobin and thermosensitive TRP channels. We suggest this mechanism also defines basal body temperatures in homeotherms, with TW=36.32 degrees C for humans. We demonstrate that human (mammal) and chicken (Aves) body temperatures align with specific reference temperatures, and correlate with pronounced transitions at TC in hemoglobin oxygen saturation. This suggests evolutionary adaptations in homeotherms involve an interplay between oxygen supply and water's rotational transition temperatures. The IWQ-model states that proteins sense and water sets critical physiological temperatures.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"108 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178177","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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.09.611998
Alexander D Corbett, David Horsell, Taylor Watters, Shahrum Ghasemi, Lewis Henderson, Sharika Mohanan, Caroline Mullenbroich, Gil Bub, Francis Burton, Godfrey L Smith
We apply a novel microscope architecture, the Exeter Multiscope, to the problem of acquiring image data in rapid succession from nine wells of a 96 well plate. We demonstrate that the new microscope can detect contraction in cardiomyocyte monolayers which have been plated into these wells. Each well is sampled using 500 x 500 pixels across a 1.4 x 1.4 mm field of view, acquired in three colours at 3.7 Hz per well. The use of multiple illumination wavelengths provides post-hoc focus selection, further increasing the level of automation. The performance of the Exeter Multiscope is benchmarked against industry standard methods using a commercial microscope with a motorised stage and demonstrates that the Multiscope can acquire data almost 40 times faster. The data from both Multiscope and the commercial systems are processed by a 'pixel variance' algorithm that uses information from the pixel value variability over time to determine the timing and amplitude of tissue contraction. This algorithm is also benchmarked against an existing algorithm that employs an absolute difference measure of tissue contraction.
{"title":"Automated measurement of cardiomyocyte monolayer contraction using the Exeter Multiscope","authors":"Alexander D Corbett, David Horsell, Taylor Watters, Shahrum Ghasemi, Lewis Henderson, Sharika Mohanan, Caroline Mullenbroich, Gil Bub, Francis Burton, Godfrey L Smith","doi":"10.1101/2024.09.09.611998","DOIUrl":"https://doi.org/10.1101/2024.09.09.611998","url":null,"abstract":"We apply a novel microscope architecture, the Exeter Multiscope, to the problem of acquiring image data in rapid succession from nine wells of a 96 well plate. We demonstrate that the new microscope can detect contraction in cardiomyocyte monolayers which have been plated into these wells. Each well is sampled using 500 x 500 pixels across a 1.4 x 1.4 mm field of view, acquired in three colours at 3.7 Hz per well. The use of multiple illumination wavelengths provides post-hoc focus selection, further increasing the level of automation. The performance of the Exeter Multiscope is benchmarked against industry standard methods using a commercial microscope with a motorised stage and demonstrates that the Multiscope can acquire data almost 40 times faster. The data from both Multiscope and the commercial systems are processed by a 'pixel variance' algorithm that uses information from the pixel value variability over time to determine the timing and amplitude of tissue contraction. This algorithm is also benchmarked against an existing algorithm that employs an absolute difference measure of tissue contraction.","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":"142178211","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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.09.611993
Dimitrios Kolokouris, Iris Elpida Kalederoglou, Anna L Duncan, Robin A. Corey, Mark Sansom, Antonios Kolocouris
The proton-conducting domain of the influenza A M2 homotetrameric channel (M2TM-AH; residues 22-62), consisting of four transmembrane (TM; residues 22-46) and four amphipathic helices (AHs; residues 47-62), promotes the release of viral RNA via acidification. Previous studies have also proposed the formation of clusters of M2 channels in the budding neck areas in raft-like domains of the plasma membrane, 1,2 which are rich in cholesterol, resulting in cell membrane scission and viral release. Experiments showed that cholesterol has a significant contribution to lipid bilayer undulations in viral buds suggesting a significant role for cholesterol in the budding process. However, a clear explanation of membrane curvature effect based on the distribution of cholesterol around M2TM-AH clusters is lacking. Using coarse-grained molecular dynamics simulations of M2TM-AH in bilayers, we observed that M2 channels form specific clusters with conical shapes, driven by attraction of their amphipathic helices (AHs). We showed that cholesterol stabilized the formation of M2 channel clusters, by filling and bridging the conical gap between M2 channels at specific sites in the N-terminals of adjacent channels or via the C-terminal region of TM and AHs, the latter sites displaying longer interaction time and higher stability. Potential of mean force calculations showed that when cholesterols occupy the identified interfacial binding sites between two M2 channels, the dimer is stabilized by 11 kJ/mol. This translates to the cholesterol-bound dimer being populated by almost two orders of magnitude compared to a dimer lacking cholesterol. We demonstrated that the cholesterol bridged M2 channels can exert lateral force on the surrounding membrane to induce the necessary negative Gaussian curvature profile which permits the spontaneous scission of the catenoid membrane neck and leads to viral buds and scission.
{"title":"The Role of Cholesterol in M2 Clustering and Viral Budding Explained","authors":"Dimitrios Kolokouris, Iris Elpida Kalederoglou, Anna L Duncan, Robin A. Corey, Mark Sansom, Antonios Kolocouris","doi":"10.1101/2024.09.09.611993","DOIUrl":"https://doi.org/10.1101/2024.09.09.611993","url":null,"abstract":"The proton-conducting domain of the influenza A M2 homotetrameric channel (M2TM-AH; residues 22-62), consisting of four transmembrane (TM; residues 22-46) and four amphipathic helices (AHs; residues 47-62), promotes the release of viral RNA via acidification. Previous studies have also proposed the formation of clusters of M2 channels in the budding neck areas in raft-like domains of the plasma membrane, 1,2 which are rich in cholesterol, resulting in cell membrane scission and viral release. Experiments showed that cholesterol has a significant contribution to lipid bilayer undulations in viral buds suggesting a significant role for cholesterol in the budding process. However, a clear explanation of membrane curvature effect based on the distribution of cholesterol around M2TM-AH clusters is lacking. Using coarse-grained molecular dynamics simulations of M2TM-AH in bilayers, we observed that M2 channels form specific clusters with conical shapes, driven by attraction of their amphipathic helices (AHs). We showed that cholesterol stabilized the formation of M2 channel clusters, by filling and bridging the conical gap between M2 channels at specific sites in the N-terminals of adjacent channels or via the C-terminal region of TM and AHs, the latter sites displaying longer interaction time and higher stability. Potential of mean force calculations showed that when cholesterols occupy the identified interfacial binding sites between two M2 channels, the dimer is stabilized by 11 kJ/mol. This translates to the cholesterol-bound dimer being populated by almost two orders of magnitude compared to a dimer lacking cholesterol. We demonstrated that the cholesterol bridged M2 channels can exert lateral force on the surrounding membrane to induce the necessary negative Gaussian curvature profile which permits the spontaneous scission of the catenoid membrane neck and leads to viral buds and scission.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178212","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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.09.612021
Eslam Elhanafy, Amin Akbari Ahangar, Rebecca Roth, Tamer M Gamal El-Din, John R Bankston, Jing Li
Voltage-gated sodium (Nav) channels are pivotal for cellular signaling and mutations in Nav channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function (GoF), loss-of-function (LoF) effects, or both. However, the mechanism behind this functional divergence of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino-acid substitutions at equivalent gating-charge positions in VSDI and VSDII of the cardiac sodium channel Nav1.5 show differential gating-property impacts in electrophysiology measurements. We conducted 120 μs molecular dynamics (MD) simulations on wild-type and six mutants to elucidate the structural basis of their differential impacts. Our μs-scale MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis elucidated how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study represents a significant leap forward in understanding structure-function relationships in Nav channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel-related disorders.
{"title":"Elucidating the Differential Impacts of Equivalent Gating-Charge Mutations in Voltage-Gated Sodium Channels","authors":"Eslam Elhanafy, Amin Akbari Ahangar, Rebecca Roth, Tamer M Gamal El-Din, John R Bankston, Jing Li","doi":"10.1101/2024.09.09.612021","DOIUrl":"https://doi.org/10.1101/2024.09.09.612021","url":null,"abstract":"Voltage-gated sodium (Na<sub>v</sub>) channels are pivotal for cellular signaling and mutations in Na<sub>v</sub> channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function (GoF), loss-of-function (LoF) effects, or both. However, the mechanism behind this functional divergence of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino-acid substitutions at equivalent gating-charge positions in VSD<sub>I</sub> and VSD<sub>II</sub> of the cardiac sodium channel Nav1.5 show differential gating-property impacts in electrophysiology measurements. We conducted 120 μs molecular dynamics (MD) simulations on wild-type and six mutants to elucidate the structural basis of their differential impacts. Our μs-scale MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis elucidated how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study represents a significant leap forward in understanding structure-function relationships in Nav channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel-related disorders.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"48 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178179","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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.09.612121
Katherine Morelli, Sandro M Meier, Angela Zhao, Madhurima Choudhury, M Willis, Yves Barral, Jackie Vogel
The energy-consuming dynamic instability of microtubules generates significant forces which are thought to be harnessed to move large cargos in cells. However, identification of mechanisms which can capture the force released during microtubule depolymerization to move large loads has been elusive. In this work we show that a biomolecular condensate provides an elegant solution to this problem. Using live cell super-resolution microscopy, we directly observe that budding yeast +TIP bodies are nanoscale droplets with classic fluid-like behaviors which accumulate type V myosin (Myo2) at their surfaces. We find that conserved self-oligomerization interfaces in the protein Kar9 tune the biophysical properties of the viscoelastic +TIP body and its ability to efficiently move the mitotic spindle. Our findings introduce a paradigm for how forces generated by microtubule dynamics are harnessed in cells and open a frontier of research on nanoscale biomolecular condensates in their native environment.
微管耗能的动态不稳定性会产生巨大的力，人们认为可以利用这些力来移动细胞中的大型载荷。然而，能够捕捉微管解聚过程中释放的力以移动大型载荷的机制一直难以确定。在这项工作中，我们发现生物分子凝聚物为这一问题提供了一个优雅的解决方案。利用活细胞超分辨率显微镜，我们直接观察到芽殖酵母 +TIP 体是具有典型流体行为的纳米级液滴，其表面聚集了 V 型肌球蛋白（Myo2）。我们发现，蛋白质 Kar9 中保守的自聚界面调整了粘弹性 +TIP 体的生物物理特性及其有效移动有丝分裂纺锤体的能力。我们的发现为如何利用微管动力学在细胞中产生的力量引入了一个范例，并为纳米级生物分子凝聚体在其原生环境中的研究开辟了一个前沿领域。
{"title":"A fluid droplet harvests the force generated by shrinking microtubules in living cells","authors":"Katherine Morelli, Sandro M Meier, Angela Zhao, Madhurima Choudhury, M Willis, Yves Barral, Jackie Vogel","doi":"10.1101/2024.09.09.612121","DOIUrl":"https://doi.org/10.1101/2024.09.09.612121","url":null,"abstract":"The energy-consuming dynamic instability of microtubules generates significant forces which are thought to be harnessed to move large cargos in cells. However, identification of mechanisms which can capture the force released during microtubule depolymerization to move large loads has been elusive. In this work we show that a biomolecular condensate provides an elegant solution to this problem. Using live cell super-resolution microscopy, we directly observe that budding yeast +TIP bodies are nanoscale droplets with classic fluid-like behaviors which accumulate type V myosin (Myo2) at their surfaces. We find that conserved self-oligomerization interfaces in the protein Kar9 tune the biophysical properties of the viscoelastic +TIP body and its ability to efficiently move the mitotic spindle. Our findings introduce a paradigm for how forces generated by microtubule dynamics are harnessed in cells and open a frontier of research on nanoscale biomolecular condensates in their native environment.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"261 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178178","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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.09.611947
Martina Oliver Huidobro, Robert G. Endres
Turing patterns are a fundamental concept in developmental biology, describing how homogeneous tissues develop into self-organized spatial patterns. However, the classical Turing mechanism, which relies on linear stability analysis, often fails to capture the complexities of real biological systems, such as multistability, non-linearities, growth, and boundary conditions. Here, we explore the impact of these factors on Turing pattern formation, contrasting linear stability analysis with numerical simulations based on a simple reaction-diffusion model, motivated by synthetic gene-regulatory pathways. We demonstrate how non-linearities introduce multistability, leading to unexpected pattern outcomes not predicted by the traditional Turing theory. The study also examines how growth and realistic boundary conditions influence pattern robustness, revealing that different growth regimes and boundary conditions can disrupt or stabilize pattern formation. Our findings are critical for understanding pattern formation in both natural and synthetic biological systems, providing insights into engineering robust patterns for applications in synthetic biology.
{"title":"Effects of multistability, absorbing boundaries and growth on Turing pattern formation","authors":"Martina Oliver Huidobro, Robert G. Endres","doi":"10.1101/2024.09.09.611947","DOIUrl":"https://doi.org/10.1101/2024.09.09.611947","url":null,"abstract":"Turing patterns are a fundamental concept in developmental biology, describing how homogeneous tissues develop into self-organized spatial patterns. However, the classical Turing mechanism, which relies on linear stability analysis, often fails to capture the complexities of real biological systems, such as multistability, non-linearities, growth, and boundary conditions. Here, we explore the impact of these factors on Turing pattern formation, contrasting linear stability analysis with numerical simulations based on a simple reaction-diffusion model, motivated by synthetic gene-regulatory pathways. We demonstrate how non-linearities introduce multistability, leading to unexpected pattern outcomes not predicted by the traditional Turing theory. The study also examines how growth and realistic boundary conditions influence pattern robustness, revealing that different growth regimes and boundary conditions can disrupt or stabilize pattern formation. Our findings are critical for understanding pattern formation in both natural and synthetic biological systems, providing insights into engineering robust patterns for applications in synthetic biology.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178209","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}
Liquid-liquid phase separation (LLPS) phenomenon plays a vital role in multiple cell biology processes, providing a mechanism to concentrate biomolecules and promote cellular reactions locally. Despite its significance in biology, there is a lack of conventional techniques suitable for studying biphasic samples in their biologically relevant form. Here, we present a label-free and non-invasive approach to characterize protein, RNA and water in biomolecular condensates termed LLPS REstricted DIFusion of INvisible speciEs (REDIFINE). Relying on diffusion NMR measurements, REDIFINE exploits the exchange dynamics between the condensed and dispersed phases to allow the determination of not only diffusion constants in both phases but also the fractions of the species, the average radius of the condensed droplets and the exchange rate between the phases. We can also access the concentration of proteins in both phases. Observing proteins, RNAs, water, and even small molecules, REDIFINE analysis allows a rapid biophysical characterization of multicomponent condensates which is important to understand their functional roles. In comparing multiple systems, REDIFINE reveals that folded RNA-binding proteins form smaller and more dynamic droplets compared to the disordered ones. In addition, REDIFINE proved to be valuable beyond LLPS for the determination of binding constants in soluble protein-RNA without the need for titration.
{"title":"LLPS REDIFINE allows the biophysical characterization of multicomponent condensates without tags or labels","authors":"Mihajlo Novakovic, Nina Han, Nina Chiara Kathe, Yinan Ni, Leonidas Emmanouilidis, Frederic H.-T. Allain","doi":"10.1101/2024.09.10.612223","DOIUrl":"https://doi.org/10.1101/2024.09.10.612223","url":null,"abstract":"Liquid-liquid phase separation (LLPS) phenomenon plays a vital role in multiple cell biology processes, providing a mechanism to concentrate biomolecules and promote cellular reactions locally. Despite its significance in biology, there is a lack of conventional techniques suitable for studying biphasic samples in their biologically relevant form. Here, we present a label-free and non-invasive approach to characterize protein, RNA and water in biomolecular condensates termed LLPS REstricted DIFusion of INvisible speciEs (REDIFINE). Relying on diffusion NMR measurements, REDIFINE exploits the exchange dynamics between the condensed and dispersed phases to allow the determination of not only diffusion constants in both phases but also the fractions of the species, the average radius of the condensed droplets and the exchange rate between the phases. We can also access the concentration of proteins in both phases. Observing proteins, RNAs, water, and even small molecules, REDIFINE analysis allows a rapid biophysical characterization of multicomponent condensates which is important to understand their functional roles. In comparing multiple systems, REDIFINE reveals that folded RNA-binding proteins form smaller and more dynamic droplets compared to the disordered ones. In addition, REDIFINE proved to be valuable beyond LLPS for the determination of binding constants in soluble protein-RNA without the need for titration.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178176","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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.09.612142
Shoyab Ansari, Dominique Lagasca, Rania Dumarieh, Yiling Xiao, Sakshi Krishna, Yang Li, Kendra K Frederick
Amyloid forms of α-synuclein adopt different conformations depending on environmental conditions. Advances in structural biology have accelerated fibril characterization. However, it remains unclear which conformations predominate in biological settings because current methods typically not only require isolating fibrils from their native environments, but they also do not provide insight about flexible regions. To address this, we characterized α-syn amyloid seeds and used sensitivity enhanced nuclear magnetic resonance to investigate the amyloid fibrils resulting from seeded amyloid propagation in different settings. We found that the amyloid fold and conformational preferences of flexible regions are faithfully propagated in vitro and in cellular lysates. However, seeded propagation of amyloids inside cells led to the minority conformation in the seeding population becoming predominant and more ordered, and altered the conformational preferences of flexible regions. The examination of the entire ensemble of protein conformations in biological settings that is made possible with this approach may advance our understanding of protein misfolding disorders and facilitate structure-based drug design efforts.
{"title":"In cell NMR reveals cells selectively amplify and structurally remodel amyloid fibrils","authors":"Shoyab Ansari, Dominique Lagasca, Rania Dumarieh, Yiling Xiao, Sakshi Krishna, Yang Li, Kendra K Frederick","doi":"10.1101/2024.09.09.612142","DOIUrl":"https://doi.org/10.1101/2024.09.09.612142","url":null,"abstract":"Amyloid forms of α-synuclein adopt different conformations depending on environmental conditions. Advances in structural biology have accelerated fibril characterization. However, it remains unclear which conformations predominate in biological settings because current methods typically not only require isolating fibrils from their native environments, but they also do not provide insight about flexible regions. To address this, we characterized α-syn amyloid seeds and used sensitivity enhanced nuclear magnetic resonance to investigate the amyloid fibrils resulting from seeded amyloid propagation in different settings. We found that the amyloid fold and conformational preferences of flexible regions are faithfully propagated in vitro and in cellular lysates. However, seeded propagation of amyloids inside cells led to the minority conformation in the seeding population becoming predominant and more ordered, and altered the conformational preferences of flexible regions. The examination of the entire ensemble of protein conformations in biological settings that is made possible with this approach may advance our understanding of protein misfolding disorders and facilitate structure-based drug design efforts.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178180","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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.09.612101
Ethan Lee, Sarah Rauscher
The main protease (MPro) of SARS-CoV-2 is essential for viral replication and is, therefore, an important drug target. Here, we investigate two flexible loops in MPro that play a role in catalysis. Using all-atom molecular dynamics simulations, we analyze the structural ensemble of MPro in an apo state and substrate-bound state. We find that the flexible loops can adopt open, intermediate (partly open) and closed conformations in solution, which differs from the partially closed state observed in crystal structures of MPro. When the loops are in closed or intermediate states, the catalytic residues are more likely to be in close proximity, which is crucial for catalysis. Additionally, we find that substrate binding to one protomer of the homodimer increases the frequency of intermediate states in the bound protomer, while also affecting the structural propensity of the apo protomer's flexible loops. Using dynamic network analysis, we identify multiple allosteric pathways connecting the two active sites of the homodimer. Common to these pathways is an allosteric hotspot involving the N-terminus, a critical region that comprises part of the binding pocket. Taken together, the results of our simulation study provide detailed insight into the relationships between the flexible loops and substrate binding in a prime drug target for COVID-19.
SARS-CoV-2 的主要蛋白酶（MPro）对病毒复制至关重要，因此是一个重要的药物靶点。在这里，我们研究了 MPro 中在催化过程中发挥作用的两个柔性环。通过全原子分子动力学模拟，我们分析了 MPro 在无底物状态和底物结合状态下的结构组合。我们发现，柔性环在溶液中可以采用开放、中间（部分开放）和封闭构象，这与在 MPro 晶体结构中观察到的部分封闭状态不同。当环路处于闭合或中间状态时，催化残基更有可能靠近，这对催化作用至关重要。此外，我们发现底物与同源二聚体的一个原体结合会增加结合原体的中间状态频率，同时也会影响 apo 原体柔性环路的结构倾向。通过动态网络分析，我们确定了连接同源二聚体两个活性位点的多种异构途径。这些途径的共同点是涉及 N 端的异构热点，N 端是一个关键区域，包含结合口袋的一部分。总之，我们的模拟研究结果提供了对 COVID-19 主要药物靶点中柔性环和底物结合之间关系的详细见解。
{"title":"The Conformational Space of the SARS-CoV-2 Main Protease Active Site Loops is Determined by Ligand Binding and Interprotomer Allostery","authors":"Ethan Lee, Sarah Rauscher","doi":"10.1101/2024.09.09.612101","DOIUrl":"https://doi.org/10.1101/2024.09.09.612101","url":null,"abstract":"The main protease (MPro) of SARS-CoV-2 is essential for viral replication and is, therefore, an important drug target. Here, we investigate two flexible loops in MPro that play a role in catalysis. Using all-atom molecular dynamics simulations, we analyze the structural ensemble of MPro in an apo state and substrate-bound state. We find that the flexible loops can adopt open, intermediate (partly open) and closed conformations in solution, which differs from the partially closed state observed in crystal structures of MPro. When the loops are in closed or intermediate states, the catalytic residues are more likely to be in close proximity, which is crucial for catalysis. Additionally, we find that substrate binding to one protomer of the homodimer increases the frequency of intermediate states in the bound protomer, while also affecting the structural propensity of the apo protomer's flexible loops. Using dynamic network analysis, we identify multiple allosteric pathways connecting the two active sites of the homodimer. Common to these pathways is an allosteric hotspot involving the N-terminus, a critical region that comprises part of the binding pocket. Taken together, the results of our simulation study provide detailed insight into the relationships between the flexible loops and substrate binding in a prime drug target for COVID-19.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"158 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178174","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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.10.612234
Hyojun Kim, Baptiste Alric, Nolan Chan, Julien Roul, Morgan Delarue
Cells that proliferate in confined environments develop mechanical compressive stress, referred to as growth-induced pressure, which inhibits growth and division across various organisms. Recent studies have shown that in these confined spaces, the diffusivity of intracellular nanoparticles decreases. However, the physical mechanisms behind this reduction remain unclear. In this study, we use quantitative phase imaging to measure the refractive index and dry mass density of Saccharomyces cerevisiae cells proliferating under confinement in a microfluidic bioreactor. Our results indicate that the observed decrease in diffusivity can be at least attributed to the intracellular accumulation of macromolecules. Furthermore, the linear scaling between cell content and growth-induced pressure suggests that the concentrations of macromolecules and osmolytes are maintained proportionally under such pressure in S. cerevisiae.
{"title":"Intracellular dry mass density increases under growth-induced pressure","authors":"Hyojun Kim, Baptiste Alric, Nolan Chan, Julien Roul, Morgan Delarue","doi":"10.1101/2024.09.10.612234","DOIUrl":"https://doi.org/10.1101/2024.09.10.612234","url":null,"abstract":"Cells that proliferate in confined environments develop mechanical compressive stress, referred to as growth-induced pressure, which inhibits growth and division across various organisms. Recent studies have shown that in these confined spaces, the diffusivity of intracellular nanoparticles decreases. However, the physical mechanisms behind this reduction remain unclear. In this study, we use quantitative phase imaging to measure the refractive index and dry mass density of Saccharomyces cerevisiae cells proliferating under confinement in a microfluidic bioreactor. Our results indicate that the observed decrease in diffusivity can be at least attributed to the intracellular accumulation of macromolecules. Furthermore, the linear scaling between cell content and growth-induced pressure suggests that the concentrations of macromolecules and osmolytes are maintained proportionally under such pressure in S. cerevisiae.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"108 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178210","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}
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