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The cross-correlation-based analysis to digest the conformational dynamics of the mitoBK channels in terms of their modulation by flavonoids 基于互相关的分析,以消化mitoBK通道在黄酮类化合物调节方面的构象动力学。
IF 2 4区 生物学 Q3 BIOPHYSICS Pub Date : 2023-06-30 DOI: 10.1007/s00249-023-01666-9
Agata Wawrzkiewicz-Jałowiecka, Paulina Trybek, Beata Dworakowska, Piotr Bednarczyk, Przemysław Borys

The activity of mitochondrial large-conductance voltage- and (Ca^{2+})-activated (K^+) channels (mitoBK) is regulated by a number of biochemical factors, including flavonoids. In particular, naringenin (Nar) and quercetin (Que) reached reasonable scientific attention due to their well-pronounced channel-activating effects. The open-reinforcing outcomes of Nar and Que on the mitoBK channel gating have been already reported. Nevertheless, the molecular picture of the corresponding channel–ligand interactions remains still to be revealed. In this work, we investigate the effects of the Nar and Que on the conformational dynamics of the mitoBK channel. In this aim, the cross-correlation-based analysis of the single-channel signals recorded by the patch-clamp method is performed. The obtained results in the form of phase space diagrams enable us to visually monitor the effects exerted by the considered flavonoids at the level of temporal characteristics of repetitive sequences of channel conformations. It turns out that the mitoBK channel activation by naringenin and quercetin does not lead to the change in the number of clusters within the phase space diagrams, which can be related to the constant number of available channel macroconformations regardless of the flavonoid administration. The localization and occupancy of the clusters of cross-correlated sequences suggest that mitoBK channel stimulation by flavonoids affects the relative stability of channel conformations and the kinetics of switching between them. For most clusters, greater net effects are observed in terms of quercetin administration in comparison with naringenin. It indicates stronger channel interaction with Que than Nar.

线粒体大电导电压和[公式:见正文]激活的[公式:参见正文]通道(mitoBK)的活性受多种生物化学因子的调节,包括黄酮类化合物。特别是柚皮素(Nar)和槲皮素(Que)由于其显著的通道激活作用而引起了合理的科学关注。Nar和Que对mitoBK通道门控的开放增强结果已经报道。然而,相应的通道-配体相互作用的分子图谱仍有待揭示。在这项工作中,我们研究了Nar和Que对mitoBK通道构象动力学的影响。为此,对通过膜片钳方法记录的单通道信号进行基于互相关的分析。所获得的相空间图形式的结果使我们能够在通道构象重复序列的时间特征水平上直观地监测所考虑的黄酮类化合物所产生的影响。事实证明,柚皮素和槲皮素对mitoBK通道的激活不会导致相空间图中簇数的变化,这可能与可用通道大构象的恒定数量有关,而与类黄酮给药无关。交叉相关序列簇的定位和占据表明,黄酮类化合物对mitoBK通道的刺激影响通道构象的相对稳定性和它们之间转换的动力学。对于大多数聚类,与柚皮素相比,槲皮素给药的净效应更大。这表明与Que的通道交互比Nar更强。
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引用次数: 0
Choosing the right density for a concentrated protein system like gluten in a coarse-grained model 为粗粒模型中的面筋等浓缩蛋白质系统选择合适的密度。
IF 2 4区 生物学 Q3 BIOPHYSICS Pub Date : 2023-06-28 DOI: 10.1007/s00249-023-01667-8
Łukasz Mioduszewski

Large coarse-grained simulations are often conducted with an implicit solvent, which makes it hard to assess the water content of the sample and the effective concentration of the system. Here the number and the size of cavities and entanglements in the system, together with density profiles, are used to asses the homogeneity and interconnectedness of gluten. This is a continuation of an earlier article, "Viscoelastic properties of wheat gluten in a molecular dynamics study" (Mioduszewski and Cieplak 2021b). It turns out there is a wide range of densities (between 1 residue per cubic nanometer and 3 residues/nm(^3)) where the system is interconnected, but not homogeneous: there are still large empty spaces, surrounded by an entangled protein network. Those findings should be of importance to any coarse-grained simulation of large protein systems.

大型粗粒度模拟通常使用隐式溶剂进行,这使得很难评估样品的含水量和系统的有效浓度。这里,系统中空腔和缠结的数量和大小,以及密度分布,用于评估面筋的均匀性和相互连接性。这是早期文章“分子动力学研究中小麦面筋的粘弹性特性”(Mioduszewski和Cieplak 2021b)的延续。事实证明,系统相互连接但不均匀的密度范围很广(在每立方纳米1个残基到每纳米3个残基之间[公式:见正文]):仍然有很大的空位,被纠缠的蛋白质网络包围。这些发现对于任何大型蛋白质系统的粗粒度模拟都应该具有重要意义。
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引用次数: 0
Twist-stretch relations in nucleic acids 核酸中的扭转-拉伸关系。
IF 2 4区 生物学 Q3 BIOPHYSICS Pub Date : 2023-06-25 DOI: 10.1007/s00249-023-01669-6
Marco Zoli

Nucleic acids are highly deformable helical molecules constantly stretched, twisted and bent in their biological functioning. Single molecule experiments have shown that double stranded (ds)-RNA and standard ds-DNA have opposite twist-stretch patterns and stretching properties when overwound under a constant applied load. The key structural features of the A-form RNA and B-form DNA helices are here incorporated in a three-dimensional mesoscopic Hamiltonian model which accounts for the radial, bending and twisting fluctuations of the base pairs. Using path integral techniques which sum over the ensemble of the base pair fluctuations, I compute the average helical repeat of the molecules as a function of the load. The obtained twist-stretch relations and stretching properties, for short A- and B-helical fragments, are consistent with the opposite behaviors observed in kilo-base long molecules.

核酸是高度可变形的螺旋分子,在其生物功能中不断拉伸、扭曲和弯曲。单分子实验表明,双链rna和标准ds- dna在恒定负载下过度缠绕时具有相反的扭曲-拉伸模式和拉伸特性。a型RNA和b型DNA螺旋的关键结构特征在这里被纳入一个三维介观哈密顿模型,该模型解释了碱基对的径向、弯曲和扭转波动。使用对碱基对波动的集合求和的路径积分技术,我计算了分子的平均螺旋重复作为载荷的函数。对于短的A-和b -螺旋片段,得到的扭转-拉伸关系和拉伸性质与在千碱基长分子中观察到的相反行为一致。
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引用次数: 0
A model for ribosome translocation based on the alternated displacement of its subunits 基于核糖体亚基交替位移的核糖体易位模型
IF 2 4区 生物学 Q3 BIOPHYSICS Pub Date : 2023-06-09 DOI: 10.1007/s00249-023-01662-z
José S. González-García

A meaningful dilemma in ribosome translocation arising from experimental facts is that, although the ribosome–mRNA interaction force always has a significant magnitude, the ribosome still moves to the next codon on the mRNA. How does the ribosome move to the next codon in the sequence while holding the mRNA tightly? The hypothesis proposed here is that ribosome subunits alternate the grip of the ribosome on the mRNA, freeing the other subunit of such interaction for a while, thus allowing its motion to the following codon. Based on this assumption, a single-loop cycle of ribosome configurations involving the relative position of its subunits is elaborated. When its dynamic is modeled as a Markov network, it gives expressions for the average ribosome translocation speed and stall force as functions of the equilibrium constants among the proposed ribosome configurations. The calculations have a reasonable agreement with experimental results, and the succession of molecular events considered here is consistent with current biomolecular concepts of the ribosome translocation process. Thus, the alternative displacements hypothesis developed in the present work suggests a feasible explanation of ribosome translocation.

实验事实导致的核糖体易位的一个有意义的困境是,尽管核糖体- mRNA相互作用力总是具有显著的大小,但核糖体仍然移动到mRNA上的下一个密码子。核糖体如何移动到序列中的下一个密码子,同时紧紧握住mRNA ?这里提出的假设是,核糖体亚基交替控制核糖体对mRNA的控制,使另一个亚基暂时摆脱这种相互作用,从而允许其移动到下一个密码子。基于这一假设,核糖体构型的单环循环涉及其亚基的相对位置进行了阐述。将其动力学建模为马尔可夫网络时,给出了核糖体平均转运速度和失速力作为不同核糖体构型平衡常数的函数表达式。计算结果与实验结果有合理的一致性,这里考虑的分子事件的连续性与当前核糖体易位过程的生物分子概念是一致的。因此,在本工作中提出的替代置换假说为核糖体易位提供了一个可行的解释。
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引用次数: 0
Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation 密度对比分析超离心法测定可降解脂质纳米颗粒mRNA拷贝数
IF 2 4区 生物学 Q3 BIOPHYSICS Pub Date : 2023-06-08 DOI: 10.1007/s00249-023-01663-y
Alexander Bepperling, Gesa Richter

Lipid nanoparticles as delivery system for mRNA have recently attracted attention to a broader audience as COVID-19 mRNA vaccines. Their low immunogenicity and capability to deliver a variety of nucleic acids renders them an interesting and complementary alternative to gene therapy vectors like AAVs. An important quality attribute of LNPs is the copy number of the encapsulated cargo molecule. This work describes how density and molecular weight distributions obtained by density contrast sedimentation velocity can be used to calculate the mRNA copy number of a degradable lipid nanoparticle formulation. The determined average copy number of 5 mRNA molecules per LNP is consistent with the previous studies using other biophysical techniques, such as single particle imaging microscopy and multi-laser cylindrical illumination confocal spectroscopy (CICS).

脂质纳米颗粒作为mRNA的递送系统最近引起了更广泛的关注,如COVID-19 mRNA疫苗。它们的低免疫原性和传递多种核酸的能力使它们成为像aav这样的基因治疗载体的有趣和互补的替代品。LNPs的一个重要质量属性是被封装货物分子的拷贝数。这项工作描述了密度和分子量分布是如何通过密度对比沉降速度获得的,可以用来计算可降解脂质纳米颗粒配方的mRNA拷贝数。每个LNP的5个mRNA分子的平均拷贝数与先前使用其他生物物理技术(如单粒子成像显微镜和多激光圆柱照明共聚焦光谱(CICS))的研究结果一致。
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引用次数: 1
Pulling short DNA with mismatch base pairs 提取碱基对不匹配的短DNA
IF 2 4区 生物学 Q3 BIOPHYSICS Pub Date : 2023-05-30 DOI: 10.1007/s00249-023-01659-8
Navin Singh, Nehal Mathur

Due to misincorporation during gene replication, the accuracy of the gene expression is often compromised. This results in a mismatch or defective pair in the DNA molecule (James et al. 2016). Here, we present our study of the stability of DNA with defects in the thermal and force ensembles. We consider DNA with a different number of defects from 2to16 and study how the denaturation process differs in both ensembles. Using a statistical model, we calculate the melting point of the DNA chain in both the ensemble. Our findings display different manifestations of DNA denaturation in thermal and force ensembles. While the DNA with defects denatures at a lower temperature than the intact DNA, the point from which the DNA is pulled is important in force ensemble.

由于基因复制过程中的错误结合,基因表达的准确性经常受到损害。这会导致DNA分子中的不匹配或缺陷对(James et al. 2016)。在这里,我们提出了我们的DNA的稳定性与缺陷的热和力系的研究。我们考虑了从2到16的不同缺陷数的DNA,并研究了变性过程在两种集成中的差异。利用统计模型,我们计算了两个集合中DNA链的熔点。我们的研究结果显示DNA变性在热系和力系中的不同表现。虽然有缺陷的DNA比完整的DNA在较低的温度下变性,但DNA被拉出的点在力集合中是重要的。
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引用次数: 0
Comparison of three AUC techniques for the determination of the loading status and capsid titer of AAVs 三种AUC技术测定aav的负载状态和衣壳滴度的比较
IF 2 4区 生物学 Q3 BIOPHYSICS Pub Date : 2023-05-28 DOI: 10.1007/s00249-023-01661-0
Alexander Bepperling, Janine Best

Due to the rise of adeno-associated viruses (AAVs) as gene therapy delivery vectors, boundary sedimentation velocity analytical ultracentrifugation (boundary SV-AUC) has been developed into a widely used quality control assay even for release analytics. It can be considered as the “gold standard” for the determination of the loading status of empty, partially filled, and full capsids especially when conducted in multiwavelength (MWL) mode. It can be considered to provide the most accurate determination of the loading status, and it also provides information on the capsid titer, aggregates, and potential contaminants such as free DNA. MWL boundary SV-AUC can be regarded as a multi-attribute (MAM) method for the characterization of AAVs. One major drawback of the method is the high sample consumption both in terms of concentration and volume. Here, we compare two alternative AUC techniques, band SV-AUC and analytical CsCl density gradient sedimentation equilibrium AUC (CsCl SE-AUC) with the boundary SV-AUC and the MWL-SV-AUC experiment. Our data show a high consistency of the determined full/empty ratios between these techniques if the appropriate wavelengths and extinction coefficients are used.

由于腺相关病毒(aav)作为基因治疗传递载体的兴起,边界沉降速度分析超离心(边界SV-AUC)已经发展成为一种广泛使用的质量控制方法,甚至可以用于释放分析。它可以被认为是确定空、部分填充和满衣壳加载状态的“金标准”,特别是在多波长(MWL)模式下。它可以被认为提供了最准确的负载状态测定,它还提供了衣壳滴度、聚集物和潜在污染物(如游离DNA)的信息。MWL边界SV-AUC可以看作是一种多属性(MAM)表征aav的方法。该方法的一个主要缺点是在浓度和体积方面的高样品消耗。在这里,我们比较了两种可供选择的AUC技术,波段SV-AUC和分析CsCl密度梯度沉降平衡AUC (CsCl SE-AUC),以及边界SV-AUC和MWL-SV-AUC实验。我们的数据表明,如果使用适当的波长和消光系数,这些技术之间确定的满/空比具有高度一致性。
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引用次数: 3
Mechanistic insights of neuronal calcium and IP3 signaling system regulating ATP release during ischemia in progression of Alzheimer’s disease 神经元钙和IP3信号系统在阿尔茨海默病缺血过程中调节ATP释放的机制
IF 2 4区 生物学 Q3 BIOPHYSICS Pub Date : 2023-05-24 DOI: 10.1007/s00249-023-01660-1
Anand Pawar, Kamal Raj Pardasani

The mechanisms of calcium ([Ca2+]) signaling in various human cells have been widely analyzed by scientists due to its crucial role in human organs like the heartbeat, muscle contractions, bone activity, brain functionality, etc. No study is reported for interdependent [Ca2+] and IP3 mechanics regulating the release of ATP in neuron cells during Ischemia in Alzheimer’s disease advancement. In the present investigation, a finite element method (FEM) is framed to explore the interdependence of spatiotemporal [Ca2+] and IP3 signaling mechanics and its role in ATP release during Ischemia as well as in the advancement of Alzheimer’s disorder in neuron cells. The results provide us insights of the mutual spatiotemporal impacts of [Ca2+] and IP3 mechanics as well as their contributions to ATP release during Ischemia in neuron cells. The results obtained for the mechanics of interdependent systems differ significantly from the results of simple independent system mechanics and provide new information about the processes of the two systems. From this study, it is concluded that neuronal disorders cannot only be simply attributed to the disturbance caused directly in the processes of calcium signaling mechanics, but also to the disturbances caused in IP3 regulation mechanisms impacting the calcium regulation in the neuron cell and ATP release.

由于钙([Ca2+])在人体器官如心跳、肌肉收缩、骨骼活动、大脑功能等中起着至关重要的作用,科学家们广泛分析了各种人体细胞中钙([Ca2+])信号传导的机制。在阿尔茨海默病进展过程中,神经元细胞在缺血过程中相互依赖的[Ca2+]和IP3机制调节ATP的释放尚未有研究报道。在本研究中,采用有限元法(FEM)探讨时空[Ca2+]和IP3信号机制的相互依赖性及其在缺血时ATP释放和阿尔茨海默病进展中的作用。这些结果为我们提供了[Ca2+]和IP3相互时空影响的机制,以及它们对神经元细胞缺血时ATP释放的贡献。相互依赖系统力学的结果与简单的独立系统力学的结果有很大的不同,并提供了关于这两个系统过程的新信息。本研究认为,神经元疾病不能简单地归结为钙信号传导机制过程中直接受到的干扰,还可以归结为IP3调节机制受到的干扰影响了神经元细胞内钙的调节和ATP的释放。
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引用次数: 3
SDS-induced hexameric oligomerization of myotoxin-II from Bothrops asper assessed by sedimentation velocity and nuclear magnetic resonance 用沉降速度和核磁共振技术研究sds诱导的水蚤肌毒素- ii的六聚体低聚化
IF 2 4区 生物学 Q3 BIOPHYSICS Pub Date : 2023-05-20 DOI: 10.1007/s00249-023-01658-9
Amy Henrickson, Tony Montina, Paul Hazendonk, Bruno Lomonte, Ana Gisele C. Neves-Ferreira, Borries Demeler

We report the solution behavior, oligomerization state, and structural details of myotoxin-II purified from the venom of Bothrops asper in the presence and absence of sodium dodecyl sulfate (SDS) and multiple lipids, as examined by analytical ultracentrifugation and nuclear magnetic resonance. Molecular functional and structural details of the myotoxic mechanism of group II Lys-49 phospholipase A2 homologues have been only partially elucidated so far, and conflicting observations have been reported in the literature regarding the monomeric vs. oligomeric state of these toxins in solution. We observed the formation of a stable and discrete, hexameric form of myotoxin-II, but only in the presence of small amounts of SDS. In SDS-free medium, myotoxin-II was insensitive to mass action and remained monomeric at all concentrations examined (up to 3 mg/ml, 218.2 μM). At SDS concentrations above the critical micelle concentration, only dimers and trimers were observed, and at intermediate SDS concentrations, aggregates larger than hexamers were observed. We found that the amount of SDS required to form a stable hexamer varies with protein concentration, suggesting the need for a precise stoichiometry of free SDS molecules. The discovery of a stable hexameric species in the presence of a phospholipid mimetic suggests a possible physiological role for this oligomeric form, and may shed light on the poorly understood membrane-disrupting mechanism of this myotoxic protein class.

本研究报告了在十二烷基硫酸钠(SDS)和多种脂质存在和不存在的情况下,从Bothrops asper毒液中纯化的肌毒素ii的溶液行为、寡聚化状态和结构细节,并通过分析性超离心和核磁共振进行了检测。II组Lys-49磷脂酶A2同源物的肌毒性机制的分子功能和结构细节迄今为止仅部分阐明,并且关于这些毒素在溶液中的单体和寡聚状态的文献报道了相互矛盾的观察结果。我们观察到形成一个稳定的,离散的,六聚体形式的肌毒素ii,但只有在存在少量的SDS。在无sds的培养基中,myotoxin-II对质量作用不敏感,在所有检测浓度(高达3mg /ml, 218.2 μM)下都保持单体。当SDS浓度高于临界胶束浓度时,只观察到二聚体和三聚体,而在中等SDS浓度时,观察到大于六聚体的聚集体。我们发现,形成稳定的六聚体所需的SDS的量随蛋白质浓度的变化而变化,这表明需要精确的游离SDS分子的化学计量。在磷脂模拟物存在的情况下发现了一种稳定的六聚体物种,这表明这种寡聚物形式可能具有生理作用,并可能阐明这种肌毒性蛋白类的膜破坏机制。
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引用次数: 3
Comparative sedimentation equilibrium analysis of two IgG1 glycoforms: IgGCri and IgGWid IgG1两种糖型:IgGCri和IgGWid的沉降平衡比较分析
IF 2 4区 生物学 Q3 BIOPHYSICS Pub Date : 2023-05-17 DOI: 10.1007/s00249-023-01656-x
Khalil Abu Hammad, Vlad Dinu, Thomas E. MacCalman, Jacob Pattem, Margaret Goodall, Richard B. Gillis, Roy Jefferis, Stephen E. Harding

The solution properties of two different glycoforms of IgG1 (IgG1Cri and IgG1Wid) are compared using primarily sedimentation equilibrium analysis with two complementary analysis routines: SEDFIT-MSTAR and MULTISIG. IgGCri bears diantennary complex-type glycans on its Fc domain that are fully core fucosylated and partially sialylated, whilst on IgGWid, they are non-fucosylated, partially galactosylated and non-sialylated. IgGWid is also Fab glycosylated. Despite these differences, SEDFIT-MSTAR analysis shows similar weight average molar masses Mw of ~ (150 ± 5) kDa for IgGCri and ~ (154 ± 5) kDa for IgGWid and both glycoforms show evidence of the presence of a small fraction of dimer confirmed by MULTISIG analysis and also by sedimentation coefficient distributions from supportive sedimentation velocity measurements. The closeness of the sedimentation equilibrium behaviour and sedimentation coefficient distributions with a main peak sedimentation coefficient of ~ 6.4S for both glycoforms at different concentrations suggest that the different glycosylation profiles do not significantly impact on molar mass (molecular weight) nor conformation in solution.

使用主要的沉降平衡分析和两个互补的分析程序:SEDFIT-MSTAR和MULTISIG,比较了IgG1的两种不同糖型(IgG1Cri和IgG1Wid)的溶液性质。IgGCri在其Fc结构域上具有完全核心聚焦和部分唾液化的双链复合物型聚糖,而在IgGWid上,它们是非聚焦,部分半乳糖化和非唾液化。IgGWid也被Fab糖基化。尽管存在这些差异,SEDFIT-MSTAR分析显示,IgGCri和IgGWid的重量平均摩尔质量相似,分别为~(150±5)kDa和~(154±5)kDa。MULTISIG分析和支持沉降速度测量的沉降系数分布证实,这两种糖型都存在一小部分二聚体。两种糖型在不同浓度下的沉降平衡行为和沉降系数分布非常接近,沉降系数主峰为~ 6.4S,这表明不同的糖基化分布对溶液中的摩尔质量(分子量)和构象没有显著影响。
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引用次数: 1
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