Pub Date : 2024-09-15DOI: 10.1101/2024.09.14.613090
Kaixuan Gao, Xin Zhang, Jia Nie, Hengyu Meng, Weishe Zhang, Boxue Tian, Xiangyu Liu
G protein-coupled receptors (GPCRs) play pivotal roles in cellular signaling and represent prominent drug targets. Structural elucidation of GPCRs is crucial for drug discovery efforts. However, structural studies of GPCRs remain challenging, particularly for inactive state structures, which often require extensive protein engineering. Here, we present a de novo design strategy termed "click fusion" for generating fusion proteins to facilitate GPCR structural studies. Our method involves the rational design of structurally stable protein domains rigidly linked to GPCRs. The resulting fusion protein enhances the thermostability of the target GPCR and aids in determining GPCR structures via cryo-electron microscopy (cryo-EM). We further demonstrate that the designed fusion protein can be transferred among structurally similar GPCRs with minor adjustments to the linker region. Our study introduces a promising approach for facilitating GPCR structural studies and advancing drug discovery efforts.
{"title":"De novo Design of A Fusion Protein Tool for GPCR Research","authors":"Kaixuan Gao, Xin Zhang, Jia Nie, Hengyu Meng, Weishe Zhang, Boxue Tian, Xiangyu Liu","doi":"10.1101/2024.09.14.613090","DOIUrl":"https://doi.org/10.1101/2024.09.14.613090","url":null,"abstract":"G protein-coupled receptors (GPCRs) play pivotal roles in cellular signaling and represent prominent drug targets. Structural elucidation of GPCRs is crucial for drug discovery efforts. However, structural studies of GPCRs remain challenging, particularly for inactive state structures, which often require extensive protein engineering. Here, we present a de novo design strategy termed \"click fusion\" for generating fusion proteins to facilitate GPCR structural studies. Our method involves the rational design of structurally stable protein domains rigidly linked to GPCRs. The resulting fusion protein enhances the thermostability of the target GPCR and aids in determining GPCR structures via cryo-electron microscopy (cryo-EM). We further demonstrate that the designed fusion protein can be transferred among structurally similar GPCRs with minor adjustments to the linker region. Our study introduces a promising approach for facilitating GPCR structural studies and advancing drug discovery efforts.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"75 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255307","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 : 2024-09-14DOI: 10.1101/2024.08.15.607948
Emanuele Zippo, Dorothee Dormann, Thomas Speck, Lukas Stelzl
Understanding the condensation and aggregation of intrinsically disordered proteins in a non-equilibrium environment is crucial for unraveling many biological processes. Active enzymes catalyse many processes by consuming chemical fuels such as ATP. Enzymes called kinases phosphorylate disordered regions of proteins and thus profoundly affect their properties and interactions. Protein phosphorylation is implicated in neurodegenerative diseases and may modulate pathogenesis. However, how protein sequence and molecular recognition of a disordered protein by kinases determine phosphorylation patterns is not understood. In principle, molecular dynamics simulations hold the promise to resolve how phosphorylation affects disordered proteins and their assemblies. In practice, chemically-detailed simulations of enzymatic reactions and the dynamics of enzymes are highly challenging, in particular it is difficult to verify whether implementations of driven simulations are thermodynamically consistent. We can now address this problem with residue-level coarse-grained molecular dynamics simulations, integrating Metropolis Monte Carlo steps to model chemical reactions. Importantly, we show how to verify by Markov-state modeling that the realisation of a non-equilibrium steady state satisfies local-detailed balance. We investigate TDP-43 phosphorylation by the kinase CK1δ in simulations, examining patterns of phosphorylation and assessing its preventive role in chain aggregation, which may be a cytoprotective mechanism in neurodegenerative diseases. We find that the degree of residue phosphorylation is determined by sequence preference and charges, rather than by the position in the chain. The phosphorylation frequency is also affected by the phosphorylation patterns, since the interactions between CK1δ and TDP-43 actively change after each reaction. For TDP-43, our simulations show condensates dissolution through phosphorylation with kinases binding to the condensates and phosphorylating TDP-43 in the condensates.
{"title":"Molecular simulations of enzymatic phosphorylation of disordered proteins and their condensates","authors":"Emanuele Zippo, Dorothee Dormann, Thomas Speck, Lukas Stelzl","doi":"10.1101/2024.08.15.607948","DOIUrl":"https://doi.org/10.1101/2024.08.15.607948","url":null,"abstract":"Understanding the condensation and aggregation of intrinsically disordered proteins in a non-equilibrium environment is crucial for unraveling many biological processes. Active enzymes catalyse many processes by consuming chemical fuels such as ATP. Enzymes called kinases phosphorylate disordered regions of proteins and thus profoundly affect their properties and interactions. Protein phosphorylation is implicated in neurodegenerative diseases and may modulate pathogenesis. However, how protein sequence and molecular recognition of a disordered protein by kinases determine phosphorylation patterns is not understood. In principle, molecular dynamics simulations hold the promise to resolve how phosphorylation affects disordered proteins and their assemblies. In practice, chemically-detailed simulations of enzymatic reactions and the dynamics of enzymes are highly challenging, in particular it is difficult to verify whether implementations of driven simulations are thermodynamically consistent. We can now address this problem with residue-level coarse-grained molecular dynamics simulations, integrating Metropolis Monte Carlo steps to model chemical reactions. Importantly, we show how to verify by Markov-state modeling that the realisation of a non-equilibrium steady state satisfies local-detailed balance. We investigate TDP-43 phosphorylation by the kinase CK1δ in simulations, examining patterns of phosphorylation and assessing its preventive role in chain aggregation, which may be a cytoprotective mechanism in neurodegenerative diseases. We find that the degree of residue phosphorylation is determined by sequence preference and charges, rather than by the position in the chain. The phosphorylation frequency is also affected by the phosphorylation patterns, since the interactions between CK1δ and TDP-43 actively change after each reaction. For TDP-43, our simulations show condensates dissolution through phosphorylation with kinases binding to the condensates and phosphorylating TDP-43 in the condensates.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"69 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255357","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 : 2024-09-14DOI: 10.1101/2024.09.12.612290
Iryna Oliynyk
The use of inert gases in biology and medicine and their effect on biological objects both in vitro and in vivo remains an active area of research. It has been established that light noble gases affect antioxidant processes, free radical oxidation, and enhance chemiluminescence, but an explanation of the physical and chemical mechanisms of this effect is still lacking and is key to further theoretical and experimental studies, given the broad prospects for the use of noble gases in medicine. In this article, we present two of the possible mechanisms of light inert gases' effect on chemiluminescence (CL), a phenomenon that occurs as a result of free radical recombination and chain breakage during lipid peroxidation. Since the effect on oxidation, in turn, precedes the effect on the antioxidant system and the body's defense mechanisms. One of the mechanisms of influence is based on the ability of inert gases to dissolve well in lipids and dissolve poorly in water. Their ability to dissolve in lipid bilayers and affect the conformation of lipid complexes can increase the surface area available for oxidation, the surface area that absorbs radiation and reduce the density of the environment, potentially increasing the availability of oxygen for oxidation reactions. This is the so-called spatial mechanism of inert gas influence on oxidation and chemiluminescence. The second mechanism is based on the influence on the quantum chemical parameters of the reaction medium. The acceleration of VT relaxation processes, the impact on the components of the medium in quenching excited states, and the radiative decay time of the excited state.
{"title":"Possible Mechanisms Of Action Of Light Inert Gases On Chemiluminescence Arising As A Result Of Lipid Peroxidation","authors":"Iryna Oliynyk","doi":"10.1101/2024.09.12.612290","DOIUrl":"https://doi.org/10.1101/2024.09.12.612290","url":null,"abstract":"The use of inert gases in biology and medicine and their effect on biological objects both in vitro and in vivo remains an active area of research. It has been established that light noble gases affect antioxidant processes, free radical oxidation, and enhance chemiluminescence, but an explanation of the physical and chemical mechanisms of this effect is still lacking and is key to further theoretical and experimental studies, given the broad prospects for the use of noble gases in medicine. In this article, we present two of the possible mechanisms of light inert gases' effect on chemiluminescence (CL), a phenomenon that occurs as a result of free radical recombination and chain breakage during lipid peroxidation. Since the effect on oxidation, in turn, precedes the effect on the antioxidant system and the body's defense mechanisms. One of the mechanisms of influence is based on the ability of inert gases to dissolve well in lipids and dissolve poorly in water. Their ability to dissolve in lipid bilayers and affect the conformation of lipid complexes can increase the surface area available for oxidation, the surface area that absorbs radiation and reduce the density of the environment, potentially increasing the availability of oxygen for oxidation reactions. This is the so-called spatial mechanism of inert gas influence on oxidation and chemiluminescence. The second mechanism is based on the influence on the quantum chemical parameters of the reaction medium. The acceleration of VT relaxation processes, the impact on the components of the medium in quenching excited states, and the radiative decay time of the excited state.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"194 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255353","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 : 2024-09-14DOI: 10.1101/2024.09.13.612646
Beatriz Fonseca, Colin L. Freeman, Matthew James Collins
It is a strange observation, given the cultural co-evolution of dairying, that milk proteins are more commonly reported than any other food proteins in the archaeological record. The whey protein β-lactoglobulin and in particular its eleven amino acid long peptide T125PEVDXEALEK135 seems to be preferentially preserved in both ceramic vessels and teeth (dental calculus). An amino acid substitution in the middle of the chain is valuable to track livestock management because it permits differentiation between key animal species used in dairying. The persistence of this peptide is, however, unusual as its acidic nature makes it more vulnerable to hydrolysis. Moreover, selection for the ability to digest raw milk - more specifically, the continued production of the milk sugar enzyme lactase beyond the age of normal weaning - did not begin until the early Bronze Age. It is therefore unclear why it is a milk peptide, in particular a peptide associated with the lactose-rich whey fraction, that is one of the most commonly recovered dietary peptides. The unexpected preservation of T125PEVDXEALEK135 thus presents a good case study to uncover patterns of protein survival in the archaeological record. We have previously explored the dynamics of the bovine variation of the peptide (X=Asp130) and its likelihood to undergo hydrolysis in solution. In this study, we turn our attention to the ovine (X=Asn130) and the caprine (X=Lys130) variations of the β-lactoglobulin peptide to determine how the mutation in the amino acid in position 6 affects peptide conformations and vulnerability in bulk water. To do this, we use Molecular Dynamics as implemented in GROMACS 2020, with the Amber14SB forcefield and the SPC/E water model. We first perform extensive conformational analysis of both peptides in solution to determine stable structures. Then, using analogous methodology to that developed in our earlier study of the bovine peptide, we identify geometric arrangements between water and peptide that may be more prone to hydrolysis.
{"title":"The Survival of β-lactoglobulin Peptides in the Archaeological Record: Vulnerability vs. Sequence Variation","authors":"Beatriz Fonseca, Colin L. Freeman, Matthew James Collins","doi":"10.1101/2024.09.13.612646","DOIUrl":"https://doi.org/10.1101/2024.09.13.612646","url":null,"abstract":"It is a strange observation, given the cultural co-evolution of dairying, that milk proteins are more commonly reported than any other food proteins in the archaeological record. The whey protein β-lactoglobulin and in particular its eleven amino acid long peptide T<sub>125</sub>PEVDXEALEK<sub>135</sub> seems to be preferentially preserved in both ceramic vessels and teeth (dental calculus). An amino acid substitution in the middle of the chain is valuable to track livestock management because it permits differentiation between key animal species used in dairying. The persistence of this peptide is, however, unusual as its acidic nature makes it more vulnerable to hydrolysis. Moreover, selection for the ability to digest raw milk - more specifically, the continued production of the milk sugar enzyme lactase beyond the age of normal weaning - did not begin until the early Bronze Age. It is therefore unclear why it is a milk peptide, in particular a peptide associated with the lactose-rich whey fraction, that is one of the most commonly recovered dietary peptides. The unexpected preservation of T<sub>125</sub>PEVDXEALEK<sub>135</sub> thus presents a good case study to uncover patterns of protein survival in the archaeological record. We have previously explored the dynamics of the bovine variation of the peptide (X=Asp<sub>130</sub>) and its likelihood to undergo hydrolysis in solution. In this study, we turn our attention to the ovine (X=Asn<sub>130</sub>) and the caprine (X=Lys<sub>130</sub>) variations of the β-lactoglobulin peptide to determine how the mutation in the amino acid in position 6 affects peptide conformations and vulnerability in bulk water. To do this, we use Molecular Dynamics as implemented in GROMACS 2020, with the Amber14SB forcefield and the SPC/E water model. We first perform extensive conformational analysis of both peptides in solution to determine stable structures. Then, using analogous methodology to that developed in our earlier study of the bovine peptide, we identify geometric arrangements between water and peptide that may be more prone to hydrolysis.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255352","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 : 2024-09-14DOI: 10.1101/2024.09.10.612267
Irfan Lone
The establishment and interpretation of the concentration distribution of the morphogen Bicoid (Bcd) is considered crucial for the successful embryonic development of fruit flies. However, the biophysical mechanisms behind the timely formation and subsequent interpretation of this prototypical morphogenetic system by its target genes are not yet completely understood. Recently a discrete time, one-dimensional quantum walk model of Bcd gradient formation has been successfully used to explain the observed multiple dynamic modes of the Bcd system. However, the question of its precise interpretation by its primary target gene hunchback (hb) remains still unanswered. In this paper it will be shown that the interpretation of the Bcd gradient by its primary target gene hb, with the observed precision of ∼ 10%, takes a time period of less than a second, as expected on the basis of recent experimental observations. Furthermore, the quantum walk model is also used to explain certain key observations of recent optogenetic experiments concerning the time windows for Bcd interpretation. Finally, it is concluded that the incorporation of quantum effects into the treatment of Bcd gradient represents a viable step in exploring the dynamics of morphogen gradients.
{"title":"Quantum mechanics predicts Bicoid interpretation times of less than a second","authors":"Irfan Lone","doi":"10.1101/2024.09.10.612267","DOIUrl":"https://doi.org/10.1101/2024.09.10.612267","url":null,"abstract":"The establishment and interpretation of the concentration distribution of the morphogen Bicoid (Bcd) is considered crucial for the successful embryonic development of fruit flies. However, the biophysical mechanisms behind the timely formation and subsequent interpretation of this prototypical morphogenetic system by its target genes are not yet completely understood. Recently a discrete time, one-dimensional quantum walk model of Bcd gradient formation has been successfully used to explain the observed multiple dynamic modes of the Bcd system. However, the question of its precise interpretation by its primary target gene hunchback (hb) remains still unanswered. In this paper it will be shown that the interpretation of the Bcd gradient by its primary target gene hb, with the observed precision of ∼ 10%, takes a time period of less than a second, as expected on the basis of recent experimental observations. Furthermore, the quantum walk model is also used to explain certain key observations of recent optogenetic experiments concerning the time windows for Bcd interpretation. Finally, it is concluded that the incorporation of quantum effects into the treatment of Bcd gradient represents a viable step in exploring the dynamics of morphogen gradients.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"213 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255355","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 : 2024-09-14DOI: 10.1101/2024.09.14.613025
Hansen Tjo, Virginia Jiang, Jerelle A Joseph, Jonathan M Conway
Sugar transport into microbial cells is a critical, yet understudied step in the conversion of lignocellulosic biomass to metabolic products. Anaerocellum bescii (formerly Caldicellulosiruptor bescii) is an extremely thermophilic, anaerobic bacterium that readily degrades the cellulose and hemicellulose components of lignocellulosic biomass into a diversity of oligosaccharide substrates. Despite significant understanding of how this microorganism degrades lignocellulose, the mechanisms underlying its highly efficient transport of the resulting oligosaccharides into the cell are comparatively underexplored. Here, we identify and characterize the ATP-Binding Cassette (ABC) transporters in A. bescii governing maltodextrin transport. Utilizing past transcriptomic studies on Anaerocellum and Caldicellulosiruptor species, we identify two maltodextrin transporters in A. bescii and express and purify their substrate-binding proteins (Athe_2310 and Athe_2574) for characterization. Using differential scanning calorimetry and isothermal titration calorimetry, we show that Athe_2310 strongly interacts with shorter maltodextrins such as maltose and trehalose with dissociation constants in the micromolar range, while Athe_2574 binds longer maltodextrins, with dissociation constants in the sub-micro molar range. Using a sequence-structure-function comparison approach combined with molecular modeling we provide context for the specificity of each of these substrate-binding proteins. We propose that A. bescii utilizes orthogonal ABC transporters to uptake malto-oligosaccharides of different lengths to maximize transport efficiency.
在木质纤维素生物质转化为代谢产物的过程中，糖分输送到微生物细胞中是一个关键步骤，但对这一步骤的研究却不够深入。Anaerocellum bescii（原名 Caldicellulosiruptor bescii）是一种嗜热性极强的厌氧细菌，它能轻易地将木质纤维素生物质中的纤维素和半纤维素成分降解为多种寡糖底物。尽管人们对这种微生物如何降解木质纤维素有了深入了解，但对其将降解后的低聚糖高效转运到细胞中的机制却相对缺乏探索。在这里，我们鉴定并描述了 A. bescii 中管理麦芽糊精转运的 ATP 结合盒（ABC）转运体。利用过去对 Anaerocellum 和 Caldicellulosiruptor 物种进行的转录组学研究，我们确定了 A. bescii 中的两种麦芽糊精转运体，并表达和纯化了它们的底物结合蛋白（Athe_2310 和 Athe_2574）以进行表征。利用差示扫描量热法和等温滴定量热法，我们发现 Athe_2310 与较短的麦芽糊精（如麦芽糖和曲哈糖）有强烈的相互作用，解离常数在微摩尔范围内；而 Athe_2574 与较长的麦芽糊精结合，解离常数在亚微摩尔范围内。我们采用序列-结构-功能比较法并结合分子建模，为这些底物结合蛋白的特异性提供了背景。我们提出，贝西虫利用正交 ABC 转运体吸收不同长度的麦芽寡糖，以最大限度地提高转运效率。
{"title":"Maltodextrin Transport in the Extremely Thermophilic, Lignocellulose Degrading Bacterium Anaerocellum bescii (f. Caldicellulosiruptor bescii)","authors":"Hansen Tjo, Virginia Jiang, Jerelle A Joseph, Jonathan M Conway","doi":"10.1101/2024.09.14.613025","DOIUrl":"https://doi.org/10.1101/2024.09.14.613025","url":null,"abstract":"Sugar transport into microbial cells is a critical, yet understudied step in the conversion of lignocellulosic biomass to metabolic products. Anaerocellum bescii (formerly Caldicellulosiruptor bescii) is an extremely thermophilic, anaerobic bacterium that readily degrades the cellulose and hemicellulose components of lignocellulosic biomass into a diversity of oligosaccharide substrates. Despite significant understanding of how this microorganism degrades lignocellulose, the mechanisms underlying its highly efficient transport of the resulting oligosaccharides into the cell are comparatively underexplored. Here, we identify and characterize the ATP-Binding Cassette (ABC) transporters in A. bescii governing maltodextrin transport. Utilizing past transcriptomic studies on Anaerocellum and Caldicellulosiruptor species, we identify two maltodextrin transporters in A. bescii and express and purify their substrate-binding proteins (Athe_2310 and Athe_2574) for characterization. Using differential scanning calorimetry and isothermal titration calorimetry, we show that Athe_2310 strongly interacts with shorter maltodextrins such as maltose and trehalose with dissociation constants in the micromolar range, while Athe_2574 binds longer maltodextrins, with dissociation constants in the sub-micro molar range. Using a sequence-structure-function comparison approach combined with molecular modeling we provide context for the specificity of each of these substrate-binding proteins. We propose that A. bescii utilizes orthogonal ABC transporters to uptake malto-oligosaccharides of different lengths to maximize transport efficiency.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255351","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 : 2024-09-13DOI: 10.1101/2024.09.11.612425
Fabian Schuhmann, Kerem Can Akkaya, Dmytro Puchkov, Martin Lehmann, Fan Liu, Weria Pezeshkian
Cross-linking mass spectrometry (XL-MS) enables the mapping of protein-protein interactions on the cellular level. When applied to all compartments of mitochondria, the sheer number of cross-links and connections can be overwhelming, rendering simple cluster analyses convoluted and uninformative. To address this limitation, we integrate the XL-MS data, 3D electron microscopy data, and localization annotations with a supra coarse-grained molecular dynamics simulation to sort all data, making clusters more accessible and interpretable. In the context of mitochondria, this method, through a total of 6.9 milliseconds of simulations, successfully identifies known, suggests unknown protein clusters, and reveals the distribution of inner mitochondrial membrane proteins allowing a more precise localization within compartments. Our integrative approach suggests, that two so-far ambigiously placed proteins FAM162A and TMEM126A are localized in the cristae, which is validated through super resolution microscopy. Together, this demonstrates the strong potential of the presented approach.
{"title":"Integrative Molecular Dynamics Simulations Untangle Cross-Linking Data to Unveil Mitochondrial Protein Distributions","authors":"Fabian Schuhmann, Kerem Can Akkaya, Dmytro Puchkov, Martin Lehmann, Fan Liu, Weria Pezeshkian","doi":"10.1101/2024.09.11.612425","DOIUrl":"https://doi.org/10.1101/2024.09.11.612425","url":null,"abstract":"Cross-linking mass spectrometry (XL-MS) enables the mapping of protein-protein interactions on the cellular level. When applied to all compartments of mitochondria, the sheer number of cross-links and connections can be overwhelming, rendering simple cluster analyses convoluted and uninformative. To address this limitation, we integrate the XL-MS data, 3D electron microscopy data, and localization annotations with a supra coarse-grained molecular dynamics simulation to sort all data, making clusters more accessible and interpretable. In the context of mitochondria, this method, through a total of 6.9 milliseconds of simulations, successfully identifies known, suggests unknown protein clusters, and reveals the distribution of inner mitochondrial membrane proteins allowing a more precise localization within compartments. Our integrative approach suggests, that two so-far ambigiously placed proteins FAM162A and TMEM126A are localized in the cristae, which is validated through super resolution microscopy. Together, this demonstrates the strong potential of the presented approach.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"47 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178154","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 : 2024-09-13DOI: 10.1101/2024.09.12.612744
Orhi Esarte Palomero, Paul G DeCaen
PKD2 is a member of the polycystin subfamily of transient receptor potential (TRP) ion channel subunits which traffic and function in primary cilia organelle membranes. Millions of individuals carry pathogenic genetic variants in PKD2 that cause a life-threatening condition called autosomal dominant polycystic kidney disease (ADPKD). Although ADPKD is a common monogenetic disorder, there is no drug cure or available therapeutics which address the underlying channel dysregulation. Furthermore, the structural and mechanistic impact of most disease-causing variants are uncharacterized. Using direct cilia electrophysiology, cryogenic electron microscopy (cryo-EM), and super resolution imaging, we have discovered mechanistic differences in channel dysregulation caused by three germline missense variants located in PKD2s pore helix 1. Variant C632R reduces protein thermal stability, resulting in impaired channel assembly and abolishes primary cilia trafficking. In contrast, variants F629S and R638C retain native cilia trafficking, but exhibit gating defects. Resolved cryo-EM structures (2.7-3.2 Angstrom) of the variants indicate loss of critical pore helix interactions and precipitate allosteric collapse of the channels inner gate. Results demonstrate how ADPKD-causing these mutations have divergent and ranging impacts on PKD2 function, despite their shared structural proximity. These unexpected findings underscore the need for mechanistic characterization of polycystin variants, which may guide rational drug development of ADPKD therapeutics.
{"title":"ADPKD variants in the PKD2 pore helix cause structural collapse of the gate and distinct forms of channel dysfunction.","authors":"Orhi Esarte Palomero, Paul G DeCaen","doi":"10.1101/2024.09.12.612744","DOIUrl":"https://doi.org/10.1101/2024.09.12.612744","url":null,"abstract":"PKD2 is a member of the polycystin subfamily of transient receptor potential (TRP) ion channel subunits which traffic and function in primary cilia organelle membranes. Millions of individuals carry pathogenic genetic variants in PKD2 that cause a life-threatening condition called autosomal dominant polycystic kidney disease (ADPKD). Although ADPKD is a common monogenetic disorder, there is no drug cure or available therapeutics which address the underlying channel dysregulation. Furthermore, the structural and mechanistic impact of most disease-causing variants are uncharacterized. Using direct cilia electrophysiology, cryogenic electron microscopy (cryo-EM), and super resolution imaging, we have discovered mechanistic differences in channel dysregulation caused by three germline missense variants located in PKD2s pore helix 1. Variant C632R reduces protein thermal stability, resulting in impaired channel assembly and abolishes primary cilia trafficking. In contrast, variants F629S and R638C retain native cilia trafficking, but exhibit gating defects. Resolved cryo-EM structures (2.7-3.2 Angstrom) of the variants indicate loss of critical pore helix interactions and precipitate allosteric collapse of the channels inner gate. Results demonstrate how ADPKD-causing these mutations have divergent and ranging impacts on PKD2 function, despite their shared structural proximity. These unexpected findings underscore the need for mechanistic characterization of polycystin variants, which may guide rational drug development of ADPKD therapeutics.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"108 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178153","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 : 2024-09-13DOI: 10.1101/2024.09.12.612735
Carmelo Tempra, Victor Cruces Chamorro, Titas Mandal, Salvatore Chiantia, Martin Vogele, Balazs Fabian, Matti Javanainen
Recent advances in hydrodynamic theory have revealed the severe effect of periodic boundary conditions (PBCs) on the diffusive dynamics of lipid membranes in molecular dynamics simulations. Even when accounting for PBC effects, the corrected lipid diffusion coefficients often severely overshoot the experimental estimates. Here, we investigate the underlying reasons for the exaggerated dynamics, and suggest potential ways for improvement. To this end, we examine the diffusion of four lipid types in both bilayers and monolayers using the CHARMM36 force field. We account for PBC effects using the full hydrodynamic treatment: for bilayers we use non-equilibrium simulations to extract the interleaflet friction parameter used in the correction; whereas monolayer hydrodynamics are treated by setting this parameter to zero. Our results suggest that the dynamics of bilayers are too fast, even if interleaflet friction is accounted for. However, the change of the water model to OPC leads to an excellent agreement with experiments. For monolayers, the dynamics with the TIP3P water model agree well with experiments, whereas they are undershot with OPC. As OPC and TIP3P differ in both shear viscosity and surface tension, we develop two new mass-scaled water models to clarify the roles of the thermodynamic and kinetic properties of the water model on lipid dynamics. Our results indicate that both of these quantities play a major role in lipid dynamics. Moreover, it seems that the accurate description of diffusion in both lipid bilayers and monolayers cannot be accounted for by changes in the water model alone, but likely also requires modifications in the lipid model.
{"title":"Challenges in the Accurate Modelling of Lipid Dynamics in Monolayers and Bilayers","authors":"Carmelo Tempra, Victor Cruces Chamorro, Titas Mandal, Salvatore Chiantia, Martin Vogele, Balazs Fabian, Matti Javanainen","doi":"10.1101/2024.09.12.612735","DOIUrl":"https://doi.org/10.1101/2024.09.12.612735","url":null,"abstract":"Recent advances in hydrodynamic theory have revealed the severe effect of periodic boundary conditions (PBCs) on the diffusive dynamics of lipid membranes in molecular dynamics simulations. Even when accounting for PBC effects, the corrected lipid diffusion coefficients often severely overshoot the experimental estimates. Here, we investigate the underlying reasons for the exaggerated dynamics, and suggest potential ways for improvement.\u0000To this end, we examine the diffusion of four lipid types in both bilayers and monolayers using the CHARMM36 force field. We account for PBC effects using the full hydrodynamic treatment: for bilayers we use non-equilibrium simulations to extract the interleaflet friction parameter used in the correction; whereas monolayer hydrodynamics are treated by setting this parameter to zero.\u0000Our results suggest that the dynamics of bilayers are too fast, even if interleaflet friction is accounted for. However, the change of the water model to OPC leads to an excellent agreement with experiments. For monolayers, the dynamics with the TIP3P water model agree well with experiments, whereas they are undershot with OPC. As OPC and TIP3P differ in both shear viscosity and surface tension, we develop two new mass-scaled water models to clarify the roles of the thermodynamic and kinetic properties of the water model on lipid dynamics. Our results indicate that both of these quantities play a major role in lipid dynamics. Moreover, it seems that the accurate description of diffusion in both lipid bilayers and monolayers cannot be accounted for by changes in the water model alone, but likely also requires modifications in the lipid model.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178152","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 : 2024-09-12DOI: 10.1101/2024.09.11.612552
Fudong Xue, Wenting He, Zuo ang Xiang, Jun Ren, Chunyan Shan, Lin Yuan, Pingyong Xu
Advancing single-frame imaging techniques beyond the diffraction limit and upgrading traditional wide-field or confocal microscopes to super-resolution (SR) capabilities are greatly sought after by biologists. While enhancing image resolution by deconvolving noise-free images is beneficial, achieving a noise-free image that maintains the distribution of signal intensity poses a challenge. We first developed a denoising method utilizing reversibly switchable fluorescent proteins through synchronized signal switching (3S). Additionally, we introduced a denoising neural network technique, 3Snet, which combines supervised and self-supervised learning using 3S denoised images as the ground truth. These approaches effectively eliminate noise while maintaining fluorescence signal distribution across camera pixels. We then implemented clear image deconvolution (CLID) on both 3S and 3Snet denoised images to develop SR techniques, named 3S-CLID and 3Snet-CLID. Notably, 3Snet-CLID boosts the resolution of single fluorescence images from wide-field and spinning-disk confocal microscopies by up to 3.9 times, achieving a spatial resolution of 65 nm, the highest in such imaging scenarios without an additional SR module and complex parameter setting. 3Snet-CLID enables dual-color single-frame live-cell imaging of various subcellular structures labeled with conventional fluorescent proteins and/or dyes, allowing observations of dynamic cellular processes. We expect that these advancements will drive innovation and uncover new insights in biology.
{"title":"Parameter-free clear image deconvolution (CLID) technique for single-frame live-cell super-resolution imaging","authors":"Fudong Xue, Wenting He, Zuo ang Xiang, Jun Ren, Chunyan Shan, Lin Yuan, Pingyong Xu","doi":"10.1101/2024.09.11.612552","DOIUrl":"https://doi.org/10.1101/2024.09.11.612552","url":null,"abstract":"Advancing single-frame imaging techniques beyond the diffraction limit and upgrading traditional wide-field or confocal microscopes to super-resolution (SR) capabilities are greatly sought after by biologists. While enhancing image resolution by deconvolving noise-free images is beneficial, achieving a noise-free image that maintains the distribution of signal intensity poses a challenge. We first developed a denoising method utilizing reversibly switchable fluorescent proteins through synchronized signal switching (3S). Additionally, we introduced a denoising neural network technique, 3Snet, which combines supervised and self-supervised learning using 3S denoised images as the ground truth. These approaches effectively eliminate noise while maintaining fluorescence signal distribution across camera pixels. We then implemented clear image deconvolution (CLID) on both 3S and 3Snet denoised images to develop SR techniques, named 3S-CLID and 3Snet-CLID. Notably, 3Snet-CLID boosts the resolution of single fluorescence images from wide-field and spinning-disk confocal microscopies by up to 3.9 times, achieving a spatial resolution of 65 nm, the highest in such imaging scenarios without an additional SR module and complex parameter setting. 3Snet-CLID enables dual-color single-frame live-cell imaging of various subcellular structures labeled with conventional fluorescent proteins and/or dyes, allowing observations of dynamic cellular processes. We expect that these advancements will drive innovation and uncover new insights in biology.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178156","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}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：