Biophysics of Large Membrane Channels

大膜通道的生物物理学

基本信息

批准号：
9550253
负责人：
sergey bezrukov
金额：
$ 118.9万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9550253
关键词：
Agreement Alamethicin Anesthetics Anions Atherosclerosis Bacillus anthracis Bacillus brevis Bacterial Toxins Behavior Binding Biological Biological Models Biological Phenomena Biomimetics Biophysics Bromides Cations Cell Membrane Permeability Cell physiology Cells Cellular Membrane Characteristics Child Chromatography Clostridium botulinum Clostridium perfringens Clostridium perfringens epsilon toxin Confocal Microscopy Consensus Crowding Defect Dependence Disease Dissociation Egg Yolk Proteins Electrolytes Environment Epilepsy Escherichia coli Exclusion Fluorescence Goals Hemolysin Integral Membrane Protein Ion Channel Ion Transport Ions Kinetics Learning Lipid Bilayers Lipid Binding Lipids Medical Membrane Membrane Lipids Membrane Proteins Methods Microtubules Mitochondria Modeling Modernization Molecular Molecular Analysis Molecular Conformation Mono-S Motivation Mycobacterium smegmatis NBL1 gene Nature Neutrons Non-Insulin-Dependent Diabetes Mellitus Nutrient Organelles Organism Pathologic Peptides Peripheral Pharmaceutical Preparations Play Polymers Precipitation Proteins Pseudomonas aeruginosa Pseudomonas syringae Refractory Regulation Role Salts Seizures Series Sodium Chloride Specificity Spectrum Analysis Staphylococcus aureus Structure Study models Surface Techniques Theoretical Studies Time Toxin Transmembrane Transport Trichoderma Tubulin VDAC1 gene Voltage-Dependent Anion Channel Water Weight Work alpha Tubulin alpha synuclein amphiphilicity base beta barrel design deviant electric impedance ethylene glycol experimental study gramicidin A grasp interest mechanical properties mitochondrial membrane molecular dynamics nanopore nanoscale neurophysiology novel strategies physical property protein reconstitution reconstitution scaffold sensor single molecule sound sugar theories water solubility

项目摘要

I. Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes Tubulin has emerged as a highly unexpected component of mitochondrial membranes involved in regulation of membrane permeability. The discovery of this functional role has reawakened interest in the nature of the tubulinmembrane interaction to answer a new question: How does tubulin, a cytosolic protein famous for its role in microtubule structure and dynamics, come to target mitochondrial membranes? This year, using a combination of five biophysical methodssurface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations, we have studied peripheral binding of tubulin to biomimetic membranes of different lipid compositions. We find that tubulin distinguishes between lamellar and nonlamellar lipids through a highly conserved amphipathic binding motif. Specifically, alpha-tubulin targets cell and organelle membranes by sensing lipid-packing defects, with broad consequences for both normal cellular function and disease. We show that tubulin belongs to the group of peripherally bound amphitropic proteins. These proteins are the subfamily of peripheral membrane proteins that interact directly with the lipid membrane rather than with intrinsic membrane proteins and are therefore strongly influenced by lipid composition. A number of diseases, such as atherosclerosis, type II diabetes, and lysosomal storage disorders, are associated with defects in maintaining the correct distribution of intracellular lipids. Although involvement of amphitropic proteins in various cell functions is soundly established, the mechanisms of their interaction with cellular membranes are only beginning to be understood because their characteristic reversible binding to the membranes creates obvious experimental difficulties in assessing binding conformations and kinetics. II. Partitioning of soft water-soluble polymers into beta-barrel channels in their functional states Understanding polymer partitioning into nanoscale cavities of different nature is important for many technological applications that include, but are not limited to, analytical chromatography, separation techniques, and purification methods. It is also critical in the qualitative interpretation and quantitative analysis of molecular interactions and biological regulation in the crowded cellular environment. This necessitates model studies with polymer solutions explored in both dilute and semidilute regimes. Recently, we studied polymer partitioning from semidilute solutions of poly(ethylene glycol) (PEG) mixtures into a number of membrane-spanning beta-barrel channels of different origin, and the results were rationalized within the earlier formulated polymers-pushing-polymers model of nanopore partitioning. This model is based on the assumptions that the larger component of the polymer mixture, being preferentially excluded from the cavity, pushes the smaller component into the cavity, thus representing forced polymer redistribution between the bulk and the channel. This year we studied polymer mixtures by using two different methods, small-angle neutron scattering and nanopore conductance fluctuation analysis, to quantify the larger polymer parameters in the bulk and the degree of its partitioning in the pore, respectively. We first show that the reduction of the PEG 3400 characteristic size with its increasing concentration in the bulk is statistically significant but small. We then demonstrate that partitioning of the larger polymer in the nanopore is negligible if its relative weight fraction is kept under 50%, in excellent agreement with the major assumptions of the model mentioned above. These findings are important for understanding and quantifying polymer behavior in the bulk and polymer partitioning into nanopores and different protein cavities in dilute and semidilute regimes mimicking those of the crowded conditions of the cell. III. Understanding ion specificity of the Hofmeister ranking Ion specificity and, in particular, the distinctive effects of anions in salt-induced protein precipitation have been known since the 1880s, when Franz Hofmeister established the ranking of anions in their ability to regulate egg yolk protein water solubility. Experimental and theoretical studies have given a detailed empirical picture of the phenomenon, but the nature of the ionic interactions with the surfaces leading to the Hofmeister effect is still under debate. The only consensus is that it cannot be explained by standard theories of electrolytes. For example, bromide is unique in that its salts were recognized as a drug to treat epilepsy a couple of dozen years before Hofmeisters studies and they are still in use to treat specific types of refractory seizures in children, but the mechanism of their action remains elusive. Though the Hofmeister ranking of salts has been a frequent target of biological studies including channel-facilitated membrane transport, this year we decided to take advantage of arguably the simplest ion transport model of modern biophysicsthe channel formed by a linear pentadecapeptide, gramicidin A. Counterintuitively, we found that conductance of this perfectly cation-selective channel increases about twofold in the Hofmeister anion series H2PO4<Cl=Br=NO3<ClO4<SCN. Channel dissociation kinetics show even stronger dependence, with the dwell time increasing about 20-fold. While the conductance can be quantitatively explained by the changes in membrane surface potential due to exclusion of kosmotropes from (or accumulation of chaotropes at) the surface, the kinetics proved to be more difficult to treat. We estimate the effects of changes in the energetics at the bilayer surfaces on the channel dwell time, concluding that the change would have to be greater than typically observed for the Hofmeister effect outside the context of the lipid bilayer. We believe that our results are of importance for further progress in understanding of ion specificity, which manifests itself in many physicochemical and biological phenomena including the more than century-old medical applications.

I.仿生线粒体膜上小管蛋白的结构特征和脂质结合结构域微管蛋白已成为与膜通透性调节有关的线粒体膜的高度意外成分。这种功能作用的发现重新唤起了对微管蛋白膜相互作用的性质的兴趣，以回答一个新问题：微管蛋白是一种因其在微管结构和动力学中的作用而闻名的细胞质蛋白，如何靶向线粒体膜？今年，我们研究了五种生物物理方法表面等离激子共振，电化学障碍光谱，双层甲板分析，中子反射仪和分子动力学模拟的组合，我们研究了小管蛋白与生物含糊膜的外周结合。我们发现，微管蛋白通过高度保守的两亲性结合基序区分层状和非层脂质。具体而言，α-微管蛋白通过感应脂质包装缺陷来靶向细胞和细胞器膜，对正常细胞功能和疾病都有广泛的影响。我们表明微管蛋白属于周围结合的两性蛋白质。这些蛋白是直接与脂质膜而不是与固有的膜蛋白直接相互作用的外周膜蛋白的亚家族，因此受脂质组成强烈影响。许多疾病，例如动脉粥样硬化，II型糖尿病和溶酶体储存障碍，与维持细胞内脂质的正确分布的缺陷有关。尽管已经建立了两性蛋白在各种细胞功能中的参与，但它们与细胞膜相互作用的机制才开始被理解，因为它们与膜的特征可逆结合在评估结合构型和动力学方面造成了明显的实验困难。 ii。将软水溶性聚合物分配到其功能状态下的β-桶通道了解将聚合物分配到不同性质的纳米级腔中，对于包括但不限于分析色谱，分离技术和纯化方法的许多技术应用非常重要。这对于在拥挤的细胞环境中对分子相互作用和生物调节的定性解释和定量分析也至关重要。这需要在稀稀和半二元方案中探索的聚合物溶液进行模型研究。最近，我们研究了聚合物从聚（乙二醇）（PEG）混合物的半硅酸溶液中分配到不同来源的许多跨膜β-桶通道中，并且在早期配制的聚合物 - 纳米孔分析的纳米孔模型中，结果在早期配制的聚合物 - 式聚合物 - 搅拌过程中得到了合理化。该模型基于以下假设：聚合物混合物的较大分量优先排除在腔体中，将较小的成分推入腔体，从而代表了散装和通道之间的强制聚合物重新分布。今年，我们使用两种不同的方法（小角度的中子散射和纳米孔电导波动分析）研究了聚合物混合物，以分别量化大块中较大的聚合物参数及其在孔中的分配程度。我们首先表明，PEG 3400特征大小的减小，其在整体中的浓度增加具有统计学意义，但很小。然后，我们证明纳米孔中较大的聚合物的分配是可以忽略的，如果其相对重量分数保持在50％以下，并且与上述模型的主要假设非常吻合。这些发现对于理解和量化散装中的聚合物行为以及在稀释和半硫磺度方面分配到纳米孔中的聚合物行为以及模仿细胞拥挤条件的聚合物的不同蛋白腔很重要。 iii。了解HofMeister排名的离子特异性自1880年代以来，弗朗兹·霍夫迈斯特（Franz Hofmeister）在调节蛋黄蛋白水溶性的能力方面确定了阴离子的排名，尤其是离子特异性，尤其是阴离子在盐诱导的蛋白沉淀中的独特作用。实验和理论研究给出了该现象的详细经验图，但是离子相互作用与导致Hofmeister效应的表面相互作用的性质仍在争论中。唯一的共识是不能用电解质的标准理论来解释它。例如，溴是独一无二的，因为它的盐被公认为是治疗癫痫病的药物，在Hofmeisters研究之前的几十年中，它们仍在用来治疗儿童中特定类型的难治性癫痫发作，但其作用机制仍然难以捉摸。尽管盐的Hofmeister排名一直是生物学研究的常见目标，包括通道促膜的运输，今年我们决定利用现代生物物理学的最简单离子传输模型，由线性五肽肽，Gramicidin A形成的渠道，Gramicidin A.逆性性A偶发性，我们发现了该频道的完美频道，我们的电导量增加了，该频道是构成型号的范围。 H2PO4 <cl = br = no3 <clo4 <scn。通道解离动力学显示出更强的依赖性，停留时间增加了约20倍。虽然可以通过排除膜表面电位的变化来定量解释，因为将kosmotropes排除在表面上（或混合物的积累），但事实证明，动力学被证明更难治疗。我们估计了双层表面上通道停留时间的能量变化的影响，得出的结论是，在脂质双层的背景下，这种变化必须大于通常观察到的hofmeister效应的变化。我们认为，我们的结果对于在理解离子特异性方面的进一步进展至关重要，这在许多物理化学和生物学现象中都表现出了自己的表现，包括多个世纪以上的医学应用。