Pub Date : 2023-02-16DOI: 10.1007/s00249-023-01630-7
Gongyi Hong, Ruth Pachter
Although the magnetosensitivity to weak magnetic fields, such as the geomagnetic field, which was exhibited by radical pairs that are potentially responsible for avian navigation, has been previously investigated by spin dynamics simulations, understanding this behavior for proposed radical pairs in other species is limited. These include, for example, radical pairs formed in the single-cell green alga Chlamydomonas reinhardtii (CraCRY) and in Columba livia (ClCRY4). In addition, the radical pair of FADH• with the one-electron reduced cyclobutane thymine dimer that was shown to be sensitive to weak magnetic fields has been of interest. In this work, we investigated the directional magnetosensitivity of these radical pairs to a weak magnetic field by spin dynamics simulations. We find significant reduction in the magnetosensitivity by inclusion of dipolar and exchange interactions, which can be mitigated by a scavenging radical, as demonstrated for the [FAD•− TyrD•] radical pair in CraCRY, but not for the [FADH• T□T•−] radical pair because of the large exchange coupling. The directional magnetosensitivity of the ClCRY4 [FAD•− TyrE•] radical pair can survive this adverse effect even without the scavenging reaction, possibly motivating further experimental exploration.
{"title":"Effects of inter-radical interactions and scavenging radicals on magnetosensitivity: spin dynamics simulations of proposed radical pairs","authors":"Gongyi Hong, Ruth Pachter","doi":"10.1007/s00249-023-01630-7","DOIUrl":"10.1007/s00249-023-01630-7","url":null,"abstract":"<div><p>Although the magnetosensitivity to weak magnetic fields, such as the geomagnetic field, which was exhibited by radical pairs that are potentially responsible for avian navigation, has been previously investigated by spin dynamics simulations, understanding this behavior for proposed radical pairs in other species is limited. These include, for example, radical pairs formed in the single-cell green alga <i>Chlamydomonas reinhardtii</i> (<i>Cra</i>CRY) and in <i>Columba livia</i> (<i>Cl</i>CRY4). In addition, the radical pair of FADH<sup>•</sup> with the one-electron reduced cyclobutane thymine dimer that was shown to be sensitive to weak magnetic fields has been of interest. In this work, we investigated the directional magnetosensitivity of these radical pairs to a weak magnetic field by spin dynamics simulations. We find significant reduction in the magnetosensitivity by inclusion of dipolar and exchange interactions, which can be mitigated by a scavenging radical, as demonstrated for the [FAD<sup>•−</sup> TyrD<sup>•</sup>] radical pair in <i>Cra</i>CRY, but not for the [FADH<sup>•</sup> T□T<sup>•−</sup>] radical pair because of the large exchange coupling. The directional magnetosensitivity of the <i>Cl</i>CRY4 [FAD<sup>•−</sup> TyrE<sup>•</sup>] radical pair can survive this adverse effect even without the scavenging reaction, possibly motivating further experimental exploration.</p></div>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":null,"pages":null},"PeriodicalIF":2.0,"publicationDate":"2023-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4645567","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-02-15DOI: 10.1007/s00249-023-01629-0
John S. Philo
Proper interpretation of analytical ultracentrifugation (AUC) data for purified proteins requires ancillary information and calculations to account for factors such as buoyancy, buffer viscosity, hydration, and temperature. The utility program SEDNTERP has been widely used by the AUC community for this purpose since its introduction in the mid-1990s. Recent extensions to this program (1) allow it to incorporate data from diffusion as well as AUC experiments; and (2) allow it to calculate the refractive index of buffer solutions (based on the solute composition of the buffer), as well as the specific refractive increment (dn/dc) of proteins based on their composition. These two extensions should be quite useful to the light scattering community as well as helpful for AUC users. The latest version also adds new terms to the partial specific volume calculations which should improve the accuracy, particularly for smaller proteins and peptides, and can calculate the viscosity of buffers containing heavy isotopes of water. It also uses newer, more accurate equations for the density of water and for the hydrodynamic properties of rods and disks. This article will summarize and review all the equations used in the current program version and the scientific background behind them. It will tabulate the values used to calculate the partial specific volume and dn/dc, as well as the polynomial coefficients used in calculating the buffer density and viscosity (most of which have not been previously published), as well as the new ones used in calculating the buffer refractive index.
{"title":"SEDNTERP: a calculation and database utility to aid interpretation of analytical ultracentrifugation and light scattering data","authors":"John S. Philo","doi":"10.1007/s00249-023-01629-0","DOIUrl":"10.1007/s00249-023-01629-0","url":null,"abstract":"<div><p>Proper interpretation of analytical ultracentrifugation (AUC) data for purified proteins requires ancillary information and calculations to account for factors such as buoyancy, buffer viscosity, hydration, and temperature. The utility program SEDNTERP has been widely used by the AUC community for this purpose since its introduction in the mid-1990s. Recent extensions to this program (1) allow it to incorporate data from diffusion as well as AUC experiments; and (2) allow it to calculate the refractive index of buffer solutions (based on the solute composition of the buffer), as well as the specific refractive increment (<i>dn/dc</i>) of proteins based on their composition. These two extensions should be quite useful to the light scattering community as well as helpful for AUC users. The latest version also adds new terms to the partial specific volume calculations which should improve the accuracy, particularly for smaller proteins and peptides, and can calculate the viscosity of buffers containing heavy isotopes of water. It also uses newer, more accurate equations for the density of water and for the hydrodynamic properties of rods and disks. This article will summarize and review all the equations used in the current program version and the scientific background behind them. It will tabulate the values used to calculate the partial specific volume and <i>dn/dc</i>, as well as the polynomial coefficients used in calculating the buffer density and viscosity (most of which have not been previously published), as well as the new ones used in calculating the buffer refractive index.</p></div>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":null,"pages":null},"PeriodicalIF":2.0,"publicationDate":"2023-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00249-023-01629-0.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4895151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-02-14DOI: 10.1007/s00249-023-01632-5
Henry Sawczyc, Sabine Heit, Anthony Watts
From the discovery of the first membrane-interacting polymer, styrene maleic-acid (SMA), there has been a rapid development of membrane solubilising polymers. These new polymers can solubilise membranes under a wide range of conditions and produce varied sizes of nanoparticles, yet there has been a lack of broad comparison between the common polymer types and solubilising conditions. Here, we present a comparative study on the three most common commercial polymers: SMA 3:1, SMA 2:1, and DIBMA. Additionally, this work presents, for the first time, a comparative characterisation of polymethacrylate copolymer (PMA). Absorbance and dynamic light scattering measurements were used to evaluate solubilisation across key buffer conditions in a simple, adaptable assay format that looked at pH, salinity, and divalent cation concentration. Lipid-polymer nanoparticles formed from SMA variants were found to be the most susceptible to buffer effects, with nanoparticles from either zwitterionic DMPC or POPC:POPG (3:1) bilayers only forming in low to moderate salinity (< 600 mM NaCl) and above pH 6. DIBMA-lipid nanoparticles could be formed above a pH of 5 and were stable in up to 4 M NaCl. Similarly, PMA-lipid nanoparticles were stable in all NaCl concentrations tested (up to 4 M) and a broad pH range (3–10). However, for both DIBMA and PMA nanoparticles there is a severe penalty observed for bilayer solubilisation in non-optimal conditions or when using a charged membrane. Additionally, lipid fluidity of the DMPC-polymer nanoparticles was analysed through cw-EPR, showing no cooperative gel-fluid transition as would be expected for native-like lipid membranes.
自第一个膜相互作用聚合物苯乙烯马来酸(SMA)的发现以来,膜增溶聚合物得到了迅速的发展。这些新的聚合物可以在各种条件下溶解膜,并产生不同大小的纳米颗粒,然而,在常见的聚合物类型和溶解条件之间缺乏广泛的比较。在这里,我们提出了三种最常见的商业聚合物的比较研究:SMA 3:1, SMA 2:1和DIBMA。此外,这项工作首次提出了聚甲基丙烯酸酯共聚物(PMA)的比较表征。吸光度和动态光散射测量用于评估关键缓冲条件下的增溶作用,采用简单、适应性强的分析格式,观察pH、盐度和二价阳离子浓度。研究发现,由SMA变异体形成的脂质聚合物纳米颗粒最容易受到缓冲效应的影响,两性离子DMPC或POPC:POPG(3:1)双层形成的纳米颗粒仅在低至中等盐度(< 600 mM NaCl)和高于pH 6的条件下形成。dibma -脂质纳米颗粒在pH大于5的条件下可以形成,并且在高达4 M的NaCl中保持稳定。同样,pma -脂质纳米颗粒在所有测试的NaCl浓度(高达4 M)和广泛的pH范围(3-10)中都是稳定的。然而,对于DIBMA和PMA纳米粒子来说,在非最佳条件下或使用带电膜时,双分子层的增溶会受到严重的惩罚。此外,通过cw-EPR分析了dmpc -聚合物纳米颗粒的脂质流动性,显示没有天然类脂质膜所期望的凝胶-流体协同转变。
{"title":"A comparative characterisation of commercially available lipid-polymer nanoparticles formed from model membranes","authors":"Henry Sawczyc, Sabine Heit, Anthony Watts","doi":"10.1007/s00249-023-01632-5","DOIUrl":"10.1007/s00249-023-01632-5","url":null,"abstract":"<div><p>From the discovery of the first membrane-interacting polymer, styrene maleic-acid (SMA), there has been a rapid development of membrane solubilising polymers. These new polymers can solubilise membranes under a wide range of conditions and produce varied sizes of nanoparticles, yet there has been a lack of broad comparison between the common polymer types and solubilising conditions. Here, we present a comparative study on the three most common commercial polymers: SMA 3:1, SMA 2:1, and DIBMA. Additionally, this work presents, for the first time, a comparative characterisation of polymethacrylate copolymer (PMA). Absorbance and dynamic light scattering measurements were used to evaluate solubilisation across key buffer conditions in a simple, adaptable assay format that looked at pH, salinity, and divalent cation concentration. Lipid-polymer nanoparticles formed from SMA variants were found to be the most susceptible to buffer effects, with nanoparticles from either zwitterionic DMPC or POPC:POPG (3:1) bilayers only forming in low to moderate salinity (< 600 mM NaCl) and above pH 6. DIBMA-lipid nanoparticles could be formed above a pH of 5 and were stable in up to 4 M NaCl. Similarly, PMA-lipid nanoparticles were stable in all NaCl concentrations tested (up to 4 M) and a broad pH range (3–10). However, for both DIBMA and PMA nanoparticles there is a severe penalty observed for bilayer solubilisation in non-optimal conditions or when using a charged membrane. Additionally, lipid fluidity of the DMPC-polymer nanoparticles was analysed through cw-EPR, showing no cooperative gel-fluid transition as would be expected for native-like lipid membranes.</p></div>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":null,"pages":null},"PeriodicalIF":2.0,"publicationDate":"2023-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00249-023-01632-5.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4569524","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-02-14DOI: 10.1007/s00249-023-01631-6
Saeed Mortezazadeh, Borries Demeler
A method for removing time- and radially invariant noise from sedimentation velocity and sedimentation equilibrium experiments performed in an analytical ultracentrifuge is presented. The method averages repeat radial incident light measurements as a function of the photomultiplier response at different wavelengths to remove the majority of the time-invariant noise contributions from intensity data measurements. The results of this method are compared to traditional absorbance data generated with a buffer reference and the Beckman Optima AUC data acquisition program, and with the standard UltraScan refinement workflow. The method avoids the amplification of stochastic noise inherent in the absorbance scan subtraction traditionally employed in sedimentation velocity and equilibrium data. In addition, the collection of intensity data frees up the reference channel for additional samples, doubling the capacity of the instrument. In comparison to absorbance data, the residual mean square deviation of a fitted sedimentation velocity experiment without additional noise correction by UltraScan was improved by a factor of 4.5 when using the new method. This improvement benefits sedimentation equilibrium experiments as well as analytical buoyant density equilibrium experiments where routine time-invariant noise correction calculations cannot be performed.
{"title":"Systematic noise removal from analytical ultracentrifugation data with UltraScan","authors":"Saeed Mortezazadeh, Borries Demeler","doi":"10.1007/s00249-023-01631-6","DOIUrl":"10.1007/s00249-023-01631-6","url":null,"abstract":"<div><p>A method for removing time- and radially invariant noise from sedimentation velocity and sedimentation equilibrium experiments performed in an analytical ultracentrifuge is presented. The method averages repeat radial incident light measurements as a function of the photomultiplier response at different wavelengths to remove the majority of the time-invariant noise contributions from intensity data measurements. The results of this method are compared to traditional absorbance data generated with a buffer reference and the Beckman Optima AUC data acquisition program, and with the standard UltraScan refinement workflow. The method avoids the amplification of stochastic noise inherent in the absorbance scan subtraction traditionally employed in sedimentation velocity and equilibrium data. In addition, the collection of intensity data frees up the reference channel for additional samples, doubling the capacity of the instrument. In comparison to absorbance data, the residual mean square deviation of a fitted sedimentation velocity experiment without additional noise correction by UltraScan was improved by a factor of 4.5 when using the new method. This improvement benefits sedimentation equilibrium experiments as well as analytical buoyant density equilibrium experiments where routine time-invariant noise correction calculations cannot be performed.</p></div>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":null,"pages":null},"PeriodicalIF":2.0,"publicationDate":"2023-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00249-023-01631-6.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4570822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-23DOI: 10.1007/s00249-023-01628-1
Vyacheslav S. Korkosh, Denis B. Tikhonov
The gating mechanism of acid-sensitive ion channels (ASICs) remains unclear, despite the availability of atomic-scale structures in various functional states. The collapse of the acidic pocket and structural changes in the low-palm region are assumed to trigger activation. For the acidic pocket, protonation of some residues can minimize repulsion in the collapsed conformation. The relationship between low-palm rearrangements and gating is unknown. In this work, we performed a Monte Carlo energy optimization of known ASIC1a structures and determined the residue–residue interactions in different functional states. For rearrangements in the acidic pocket, our results are consistent with previously proposed mechanisms, although significant complexity was revealed for the residue–residue interactions. The data support the proposal of a gating mechanism in the low-palm region, in which residues E80 and E417 share a proton to activate the channel.
{"title":"Analysis of residue–residue interactions in the structures of ASIC1a suggests possible gating mechanisms","authors":"Vyacheslav S. Korkosh, Denis B. Tikhonov","doi":"10.1007/s00249-023-01628-1","DOIUrl":"10.1007/s00249-023-01628-1","url":null,"abstract":"<div><p>The gating mechanism of acid-sensitive ion channels (ASICs) remains unclear, despite the availability of atomic-scale structures in various functional states. The collapse of the acidic pocket and structural changes in the low-palm region are assumed to trigger activation. For the acidic pocket, protonation of some residues can minimize repulsion in the collapsed conformation. The relationship between low-palm rearrangements and gating is unknown. In this work, we performed a Monte Carlo energy optimization of known ASIC1a structures and determined the residue–residue interactions in different functional states. For rearrangements in the acidic pocket, our results are consistent with previously proposed mechanisms, although significant complexity was revealed for the residue–residue interactions. The data support the proposal of a gating mechanism in the low-palm region, in which residues E80 and E417 share a proton to activate the channel.</p></div>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":null,"pages":null},"PeriodicalIF":2.0,"publicationDate":"2023-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00249-023-01628-1.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4890356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-05DOI: 10.1007/s00249-022-01627-8
Patrick J. Fleming, John J. Correia, Karen G. Fleming
Hydration of biological macromolecules is important for their stability and function. Historically, attempts have been made to describe the degree of macromolecular hydration using a single parameter over a narrow range of values. Here, we describe a method to calculate two types of hydration: surface shell water and entrained water. A consideration of these two types of hydration helps to explain the “hydration problem” in hydrodynamics. The combination of these two types of hydration allows accurate calculation of hydrodynamic volume and related macromolecular properties such as sedimentation and diffusion coefficients, intrinsic viscosities, and the concentration-dependent non-ideality identified with sedimentation velocity experiments.
{"title":"Revisiting macromolecular hydration with HullRadSAS","authors":"Patrick J. Fleming, John J. Correia, Karen G. Fleming","doi":"10.1007/s00249-022-01627-8","DOIUrl":"10.1007/s00249-022-01627-8","url":null,"abstract":"<div><p>Hydration of biological macromolecules is important for their stability and function. Historically, attempts have been made to describe the degree of macromolecular hydration using a single parameter over a narrow range of values. Here, we describe a method to calculate two types of hydration: surface shell water and entrained water. A consideration of these two types of hydration helps to explain the “hydration problem” in hydrodynamics. The combination of these two types of hydration allows accurate calculation of hydrodynamic volume and related macromolecular properties such as sedimentation and diffusion coefficients, intrinsic viscosities, and the concentration-dependent non-ideality identified with sedimentation velocity experiments.\u0000</p></div>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":null,"pages":null},"PeriodicalIF":2.0,"publicationDate":"2023-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00249-022-01627-8.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4545147","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-02DOI: 10.1007/s00249-022-01625-w
Ivana Pajic-Lijakovic, Milan Milivojevic
Morphogenesis, tissue regeneration, and cancer invasion involve transitions in tissue morphology. These transitions, caused by collective cell migration (CCM), have been interpreted as active wetting/de-wetting transitions. This phenomenon is considered based on a model system as wetting of a cell aggregate on a rigid substrate, which includes cell aggregate movement and isotropic/anisotropic spreading of a cell monolayer around the aggregate depending on the substrate rigidity and aggregate size. This model system accounts for the transition between 3D epithelial aggregate and 2D cell monolayer as a product of: (1) tissue surface tension, (2) surface tension of substrate matrix, (3) cell–matrix interfacial tension, (4) interfacial tension gradient, (5) viscoelasticity caused by CCM, and (6) viscoelasticity of substrate matrix. These physical parameters depend on the cell contractility and state of cell–cell and cell–matrix adhesion contacts, as well as the stretching/compression of cellular systems caused by CCM. Despite extensive research devoted to study cell wetting, we still do not understand the interplay among these physical parameters which induces an oscillatory trend of cell rearrangement. This review focuses on these physical parameters in governing the cell rearrangement in the context of epithelial aggregate wetting/de-wetting, and on modeling approaches aimed at reproducing and understanding these biological systems. In this context, we not only review previously published biophysical models for cell rearrangement caused by CCM, but also propose new extensions of those models to point out the interrelation between cell–matrix interfacial tension and epithelial viscoelasticity and the role of the interfacial tension gradient in cell spreading.
{"title":"Active wetting of epithelial tissues: modeling considerations","authors":"Ivana Pajic-Lijakovic, Milan Milivojevic","doi":"10.1007/s00249-022-01625-w","DOIUrl":"10.1007/s00249-022-01625-w","url":null,"abstract":"<div><p>Morphogenesis, tissue regeneration, and cancer invasion involve transitions in tissue morphology. These transitions, caused by collective cell migration (CCM), have been interpreted as active wetting/de-wetting transitions. This phenomenon is considered based on a model system as wetting of a cell aggregate on a rigid substrate, which includes cell aggregate movement and isotropic/anisotropic spreading of a cell monolayer around the aggregate depending on the substrate rigidity and aggregate size. This model system accounts for the transition between 3D epithelial aggregate and 2D cell monolayer as a product of: (1) tissue surface tension, (2) surface tension of substrate matrix, (3) cell–matrix interfacial tension, (4) interfacial tension gradient, (5) viscoelasticity caused by CCM, and (6) viscoelasticity of substrate matrix. These physical parameters depend on the cell contractility and state of cell–cell and cell–matrix adhesion contacts, as well as the stretching/compression of cellular systems caused by CCM. Despite extensive research devoted to study cell wetting, we still do not understand the interplay among these physical parameters which induces an oscillatory trend of cell rearrangement. This review focuses on these physical parameters in governing the cell rearrangement in the context of epithelial aggregate wetting/de-wetting, and on modeling approaches aimed at reproducing and understanding these biological systems. In this context, we not only review previously published biophysical models for cell rearrangement caused by CCM, but also propose new extensions of those models to point out the interrelation between cell–matrix interfacial tension and epithelial viscoelasticity and the role of the interfacial tension gradient in cell spreading.</p></div>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":null,"pages":null},"PeriodicalIF":2.0,"publicationDate":"2023-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4083824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-12-30DOI: 10.1007/s00249-022-01626-9
Serena A. J. Watkin, Rachel Z. Bennie, Jenna M. Gilkes, Volker M. Nock, F. Grant Pearce, Renwick C. J. Dobson
Within the complex milieu of a cell, which comprises a large number of different biomolecules, interactions are critical for function. In this post-reductionist era of biochemical research, the ‘holy grail’ for studying biomolecular interactions is to be able to characterize them in native environments. While there are a limited number of in situ experimental techniques currently available, there is a continuing need to develop new methods for the analysis of biomolecular complexes that can cope with the additional complexities introduced by native-like solutions. We think approaches that use microfluidics allow researchers to access native-like environments for studying biological problems. This review begins with a brief overview of the importance of studying biomolecular interactions and currently available methods for doing so. Basic principles of diffusion and microfluidics are introduced and this is followed by a review of previous studies that have used microfluidics to measure molecular diffusion and a discussion of the advantages and challenges of this technique.
{"title":"On the utility of microfluidic systems to study protein interactions: advantages, challenges, and applications","authors":"Serena A. J. Watkin, Rachel Z. Bennie, Jenna M. Gilkes, Volker M. Nock, F. Grant Pearce, Renwick C. J. Dobson","doi":"10.1007/s00249-022-01626-9","DOIUrl":"10.1007/s00249-022-01626-9","url":null,"abstract":"<div><p>Within the complex milieu of a cell, which comprises a large number of different biomolecules, interactions are critical for function. In this post-reductionist era of biochemical research, the ‘holy grail’ for studying biomolecular interactions is to be able to characterize them in native environments. While there are a limited number of in situ experimental techniques currently available, there is a continuing need to develop new methods for the analysis of biomolecular complexes that can cope with the additional complexities introduced by native-like solutions. We think approaches that use microfluidics allow researchers to access native-like environments for studying biological problems. This review begins with a brief overview of the importance of studying biomolecular interactions and currently available methods for doing so. Basic principles of diffusion and microfluidics are introduced and this is followed by a review of previous studies that have used microfluidics to measure molecular diffusion and a discussion of the advantages and challenges of this technique.</p></div>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":null,"pages":null},"PeriodicalIF":2.0,"publicationDate":"2022-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00249-022-01626-9.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5149844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-12-22DOI: 10.1007/s00249-022-01624-x
Quan He, Shuanglan Xu, Xiaomei Ma, Ting Ling, Weiqi Feng, Xuzhi Lu, Weihua Liu, Zi Chen
Human epidermal growth factor receptor (EGFR) is involved in strong association with malignant proliferation, which has been shown to play a central role in the development and progression of non-small cell lung cancer and other solid tumors. The tumor-suppressor protein MIG6 is a negative regulator of EGFR kinase activity by binding at the activation interface of asymmetric dimer of EGFR kinase domain to disrupt EGFR dimerization and then inactivate the kinase. The protein adopts two discrete fragments 1 and 2 to directly interact with EGFR. It is revealed that the MIG6 fragment 2 is intrinsically disordered in free unbound state, but would fold into a well-structured β-hairpin when binding to EGFR, thus characterized by a so-called coupled folding-upon-binding process, which can be regarded as a compromise between favorable direct readout and unfavorable indirect readout. Here, a 23-mer F2P peptide was derived from MIG6 fragment 2, trimmed into a 17-mer tF2P peptide that contains the binding hotspot region of the fragment 2, and then constrained with an ordered hairpin conformation in free unbound state by disulfide stapling, finally resulting in a rationally stapled/trimmed stF2P peptide that largely minimizes the unfavorable indirect readout effect upon its binding to EGFR kinase domain, with affinity improved considerably upon the trimming and stapling/trimming. These rationally designed β-hairpin peptides may be further exploited as potent anti-lung cancer agents to target the activation event of EGFR dimerization.
{"title":"Coupled folding-upon-binding of human tumor suppressor MIG6 to lung cancer EGFR kinase domain and molecular trimming/stapling of MIG6-derived β-hairpins to target the coupling event","authors":"Quan He, Shuanglan Xu, Xiaomei Ma, Ting Ling, Weiqi Feng, Xuzhi Lu, Weihua Liu, Zi Chen","doi":"10.1007/s00249-022-01624-x","DOIUrl":"10.1007/s00249-022-01624-x","url":null,"abstract":"<div><p>Human epidermal growth factor receptor (EGFR) is involved in strong association with malignant proliferation, which has been shown to play a central role in the development and progression of non-small cell lung cancer and other solid tumors. The tumor-suppressor protein MIG6 is a negative regulator of EGFR kinase activity by binding at the activation interface of asymmetric dimer of EGFR kinase domain to disrupt EGFR dimerization and then inactivate the kinase. The protein adopts two discrete fragments 1 and 2 to directly interact with EGFR. It is revealed that the MIG6 fragment 2 is intrinsically disordered in free unbound state, but would fold into a well-structured β-hairpin when binding to EGFR, thus characterized by a so-called coupled folding-upon-binding process, which can be regarded as a compromise between favorable direct readout and unfavorable indirect readout. Here, a 23-mer F2P peptide was derived from MIG6 fragment 2, trimmed into a 17-mer tF2P peptide that contains the binding hotspot region of the fragment 2, and then constrained with an ordered hairpin conformation in free unbound state by disulfide stapling, finally resulting in a rationally stapled/trimmed stF2P peptide that largely minimizes the unfavorable indirect readout effect upon its binding to EGFR kinase domain, with affinity improved considerably upon the trimming and stapling/trimming. These rationally designed β-hairpin peptides may be further exploited as potent anti-lung cancer agents to target the activation event of EGFR dimerization.</p></div>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":null,"pages":null},"PeriodicalIF":2.0,"publicationDate":"2022-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4856110","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-12-13DOI: 10.1007/s00249-022-01623-y
Jeremiah N. Sims, EJun Yun, Jonathan Chu, Mansoor A. Siddiqui, Sanjay A. Desai
Intracellular calcium is maintained at very low concentrations through the action of PMCA Ca++ extrusion pumps. Although much of our knowledge about these Ca++ extrusion pumps derives from studies with human erythrocytes, kinetic studies of Ca++ transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca++ efflux using extracellular fluorescent Ca++ indicators. We optimized Ca++ loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca++ efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca++ pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca++ pump and should facilitate screens for specific inhibitors of this essential transport activity.
{"title":"A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens","authors":"Jeremiah N. Sims, EJun Yun, Jonathan Chu, Mansoor A. Siddiqui, Sanjay A. Desai","doi":"10.1007/s00249-022-01623-y","DOIUrl":"10.1007/s00249-022-01623-y","url":null,"abstract":"<div><p>Intracellular calcium is maintained at very low concentrations through the action of PMCA Ca<sup>++</sup> extrusion pumps. Although much of our knowledge about these Ca<sup>++</sup> extrusion pumps derives from studies with human erythrocytes, kinetic studies of Ca<sup>++</sup> transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca<sup>++</sup> efflux using extracellular fluorescent Ca<sup>++</sup> indicators. We optimized Ca<sup>++</sup> loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca<sup>++</sup> efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca<sup>++</sup> pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca<sup>++</sup> pump and should facilitate screens for specific inhibitors of this essential transport activity.</p></div>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":null,"pages":null},"PeriodicalIF":2.0,"publicationDate":"2022-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4822769","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}