Biophysics of Large Membrane Channels

大膜通道的生物物理学

基本信息

批准号：
8736796
负责人：
sergey bezrukov
金额：
$ 95.04万
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/8736796
关键词：
Accounting Affinity Agreement Anesthetics Anthrax disease Antidotes Bacillus anthracis Bacillus brevis Bacterial Toxins Binding Biological Biophysics Botulinum Toxins Case Study Cell Membrane Permeability Cell membrane Cell physiology Cells Characteristics Charge Chemicals Chemistry Clostridium botulinum Clostridium perfringens Clostridium perfringens epsilon toxin Complex Defect Dependence Development Diffuse Disease Dissociation Drops Drug Targeting Equilibrium Escherichia coli Fluorescence Microscopy Gases Goals Hemolysin Individual Ion Channel Label Lead Learning Lipid Bilayers Lipids Measurement Mechanics Membrane Membrane Lipids Membrane Proteins Microbial Biofilms Microtubules Mitochondria Molecular New Agents Organelles Organism Outer Mitochondrial Membrane Pattern Peptides Pharmaceutical Preparations Phenylalanine Physics Physiology Play Property Proteins Pseudomonas aeruginosa Pseudomonas syringae Publications Reaction Regulation Relative (related person)Reporting Research Respiration Role Shapes Sodium Chloride Staphylococcus aureus Structure Sum Testing Thermodynamics Time Toxin Traumatic Brain Injury Tubulin Tubulin Interaction Ultrasonography Voltage-Dependent Anion Channel Water Work amino group anthrax toxin base beta barrel beta-Cyclodextrins betadex botulinum channel blockers design deviant dioleoylphosphatidylcholine gramicidin A lens mutant novel novel strategies porin protein function protein protein interaction receptor reconstitution research study residence screening sensor simulation sugar syringomycin E theories unilamellar vesicle voltage

项目摘要

I. Interactions of high-affinity cationic blockers with the translocation pores of B. anthracis, C. botulinum, and C. perfringens binary toxins Recently, we have introduced cationic beta-cyclodextrin derivatives as highly effective and potentially universal blockers of three binary bacterial toxins: anthrax toxin of Bacillus anthracis, C2 toxin of Clostridium botulinum, and iota toxin of Clostridium perfringens. The binary toxins are made of two separate components: the enzymatic A component, which acts on certain intracellular targets, and the binding/translocation B component, which forms oligomeric channels in the target cell membrane. This year we studied the voltage and salt dependence of the rate constants of binding and dissociation reactions of two structurally different beta-cyclodextrins (AmPrbCD and AMBnTbCD) in the PA63, C2IIa, and Ib channels (B components of anthrax, C2, and iota toxins, respectively). With all three channels, the blocker carrying extra hydrophobic aromatic groups on the thio-alkyl linkers of positively charged amino groups, AMBnTbCD, demonstrated significantly stronger binding compared with AmPrbCD. This effect is seen as an increased residence time of the blocker in the channels, whereas the time between blockages characterizing the binding reaction on-rate stays practically unchanged. Surprisingly, the voltage sensitivity, expressed as a slope of the logarithm of the blocker residence time as a function of voltage, turned out to be practically the same for all six cases studied, suggesting structural similarities among the three channels. Also, the more effective AMBnTbCD blocker shows weaker salt dependence of the binding and dissociation rate constants compared with AmPrbCD. By estimating the relative contributions of the applied transmembrane field, long-range Coulomb, and salt-concentration-independent short-range forces, we found that the latter represent the leading interaction, which accounts for the high efficiency of blockage. In a search for the putative groups in the channel lumen that are responsible for the short-range forces, we performed measurements with the F427A mutant of PA63, which lacks the functionally important phenylalanine clamp. We found that the on-rates of the blockage were virtually conserved, but the residence times and, correspondingly, the binding constants dropped by more than an order of magnitude, which also reduced the difference between the efficiencies of the two blockers. Thus our quantitative analysis allowed us to discriminate between the physical forces defining the strength of interactions and, therefore, to help directed search for efficient antidotes. II. VDAC regulation by tubulin: Role of the membrane Elucidating molecular mechanisms by which lipids regulate protein function within biological membranes is critical for understanding many cellular processes. The ability of lipids to regulate proteins arises from both specific chemical features of lipid molecules and mechanical and structural properties of the lipid bilayer. Structural, compositional, and elastic parameters of lipid membranes are known to have a strong influence on the function of membrane proteins, such as ion channels, as well as on the interaction of water-soluble proteins with membranes. Recently, we have found that dimeric tubulin, a subunit of microtubules, regulates mitochondrial respiration by blocking the voltage-dependent anion channel (VDAC) of the mitochondrial outer membrane. During this reporting period, we have shown that the mechanism of VDAC blockage by tubulin involves tubulin interaction with the membrane as a critical step. The on-rate of the blockage varies up to 100-fold depending on the particular lipid composition used for bilayer formation in reconstitution experiments and increases with the increasing content of dioleoylphosphatidylethanolamine (DOPE) in dioleoylphosphatidylcholine (DOPC) bilayers. At physiologically low salt concentrations, the on-rate is decreased by the charged lipid. The off-rate of VDAC blockage by tubulin does not depend on the lipid composition. Using confocal fluorescence microscopy, we compared tubulin binding to the membranes of giant unilamellar vesicles (GUVs) made from DOPC and DOPC/ DOPE mixtures. We found that detectable binding of the fluorescently labeled dimeric tubulin to GUV membranes requires the presence of DOPE. We propose that prior to the characteristic blockage of VDAC, tubulin first binds to the membrane in a lipid-dependent manner. We thus reveal a new potent regulatory role of the mitochondrial lipids in control of the mitochondrial outer membrane permeability and hence mitochondrial respiration through tuning VDAC sensitivity to blockage by tubulin. More generally, our findings give an example of the lipid-controlled protein-protein interaction where the choice of lipid species is able to change the equilibrium binding constant by orders of magnitude. III. Physical theory of facilitated transport This year we have concentrated on two topics: (i) the effects of channel/receptor clustering and (ii) intra-membrane cavitation as a mechanism of membrane permeabilization. Various membrane functional units such as receptors, transporters, and channels, whose action necessarily involves capturing diffusing molecules, are often organized into multimeric complexes forming clusters on the cell and organelle membranes. These functional units themselves are usually oligomers of several integral proteins, which have their own symmetry. Depending on the symmetry, they form clusters on different packing lattices. Moreover, local membrane inhomogeneities, e.g., the so-called membrane domains, rafts, stalks, etc., lead to different patterns even within the structures on the same packing lattice. It is clear that the units located at the cluster periphery partially screen the units in the central part of the cluster from diffusing molecules. We were able to formulate a general approach which allows one to quantitatively describe the screening effects. The approach is used to derive simple expressions giving the trapping rates of diffusing molecules by clusters of absorbers on lattices of different packing symmetries. Our analysis shows how the trapping rate changes from the sum of the rates of individual absorbers forming the cluster to the decreasing effective collective trapping rate as the number of absorbers in the cluster increases and/or the inter-absorber distance decreases. Numerical tests demonstrate good agreement between the rates predicted by the theory and obtained from Brownian dynamics simulations for clusters of different shapes, lattice symmetries, and sizes. The study of intra-membrane cavitation (IMC) was motivated by recent publications in which it was suggested that this type of cavitation, rather than homogeneous cavitation, is crucial in ultrasound-induced membrane permeabilization and traumatic brain injury. We have focused on the thermodynamics of IMC, namely, on the minimum work required to form an intra-membrane cavity. The minimum work can be separated into two parts, one that depends on the volume and number of gas molecules in the bubble and another that depends on the bubble geometry. Using a simplified assumption about the cavity shape, the geometry-dependent term is derived and minimized at a fixed cavity volume. It was found that the optimized cavity is almost spherical at large bubble volumes, while at small volumes the cavity has a lens-like shape. The optimized shape was used to analyze the minimum work of IMC, which turned out to be significantly smaller than that of the homogeneous cavitation, thus suggesting IMC leading role in formation of membrane defects during ultrasound-induced membrane permeabilization and in traumatic brain injury.

I.高亲和力阳离子阻滞剂与炭疽芽孢杆菌，肉毒芽孢杆菌和C. perfringens二元毒素的易位孔的相互作用最近，我们引入了阳离子β-环糊精衍生物，作为三种二元细菌毒素的高效且潜在的通用阻滞剂：炭疽芽孢杆菌的炭疽毒素，肉毒乳梭状芽孢杆菌的C2毒素和裂口裂孔的Iota Toxin毒素。二进制毒素由两个单独的组件制成：酶促A成分，该成分作用于某些细胞内靶标，而结合/易位B成分，该组件在靶细胞膜中形成寡聚通道。今年，我们研究了在PA63，C2IIA和IB通道中两个结构上不同的β-环糊精（AMPRBCD和AMBNTBCD）的结合和解离反应速率常数的电压和盐依赖性（分别分别为anthrax，c2iia和ib的IB CANNENTERS（分别为anthrax，c2，c2，iota toxins的B组分）。在所有三个通道的情况下，与AmpRBCD相比，在带正电荷氨基的硫烷基接头上携带多余的疏水芳族基团的阻滞剂表现出明显更强的结合。这种效果被视为阻止器在通道中的停留时间增加，而表征结合反应上速率的阻塞时间几乎没有变化。令人惊讶的是，电压敏感性表示为阻滞剂停留时间的对数的斜率作为电压的函数，事实证明，对于所有六个研究的病例，这实际上都是相同的，这表明这三个通道之间的结构相似性。同样，与AMPRBCD相比，更有效的AMBNTBCD阻滞剂显示结合速率常数的盐依赖性较弱。通过估计应用的跨膜场，远距离库仑和盐浓度非依赖性的短距离力的相对贡献，我们发现后者代表了领先的相互作用，这说明了阻塞的高效率。在搜索负责短距离力的通道管腔中的假定组时，我们用PA63的F427A突变体进行了测量，而PA63缺乏功能上重要的苯丙氨酸夹。我们发现，封锁的率实际上是保守的，但是停留时间，相应地，结合常数降低了超过一个数量级，这也降低了两个阻滞剂的效率之间的差异。因此，我们的定量分析使我们能够区分定义相互作用强度的物理力，从而帮助定向搜索有效的解毒剂。 ii。 VDAC通过微管蛋白调节：膜的作用阐明脂质调节生物膜中蛋白质功能的分子机制对于理解许多细胞过程至关重要。脂质调节蛋白质的能力来自脂质分子的特定化学特征以及脂质双层的机械和结构特性。已知脂质膜的结构，成分和弹性参数对膜蛋白的功能（例如离子通道）以及水溶性蛋白与膜的相互作用有很大影响。最近，我们发现微管的亚基二聚体小管通过阻断线粒体外膜的电压依赖性阴离子通道（VDAC）来调节线粒体呼吸。在此报告期间，我们已经表明，微管蛋白的VDAC阻塞机理涉及与膜的相互作用作为关键步骤。堵塞的速率最高100倍，具体取决于重构实验中用于双层形成的特定脂质成分，并随着二甲酰磷脂酰甲醇胺（涂料）含量的增加而增加。在生理较低的盐浓度下，带电的脂质降低了速率。小管蛋白的VDAC阻塞的外率不取决于脂质组成。使用共聚焦荧光显微镜，我们比较了小管蛋白的结合与由DOPC和DOPC/ DOPC/ DOPE混合物制成的巨型Unilamelar囊泡（GUV）的膜。我们发现，荧光标记的二聚体小管与GUV膜的可检测结合需要涂料的存在。我们提出，在VDAC的特征阻滞之前，微管蛋白首先以脂质依赖性方式与膜结合。因此，我们揭示了线粒体脂质在控制线粒体外膜通透性中的新有效调节作用，并通过调节VDAC敏感性对微管蛋白的阻塞来调整VDAC敏感性。更笼统地，我们的发现给出了一个脂质控制蛋白 - 蛋白质相互作用的例子，其中脂质物种的选择能够通过数量级来改变平衡结合常数。 iii。促进运输的物理理论今年，我们集中在两个主题上：（i）通道/受体聚类和（ii）膜内膜的影响作为膜通透性的机理。各种膜功能单元，例如受体，转运蛋白和通道，其作用必然涉及捕获扩散的分子，通常会组织成在细胞和细胞器膜上形成簇的多聚体配合物中。这些功能单位本身通常是几种积分蛋白的低聚物，它们具有自己的对称性。根据对称性的不同，它们在不同的包装晶格上形成簇。此外，局部膜不均匀性，例如所谓的膜结构域，木筏，茎等，即使在同一包装晶格上的结构内也会产生不同的模式。显然，位于群集外围的单元部分筛选了群集中心部分的单位与扩散分子。我们能够制定一种通用方法，该方法可以定量描述筛选效果。该方法用于得出简单的表达式，从而使吸收剂簇在不同填料对称性的晶格上散布分子的捕获速率。我们的分析表明，随着群集中吸收剂的数量增加和/或吸收器间距离的降低，捕获率如何从形成簇的单个吸收器的速率变为降低有效的集体捕获率。数值测试表明，该理论预测的速率与从布朗动力学模拟获得的速率之间有着良好的一致性，用于不同形状，晶格对称和大小的簇。近期出版物的动机是针对超声诱导的膜通透性和创伤性脑损伤至关重要的，该研究的动机是由最近出版物进行的。我们专注于IMC的热力学，即形成膜内腔所需的最低工作。最低工作可以分为两个部分，一个部分取决于气泡中气体分子的体积和数量，而另一部分取决于气泡几何形状。使用关于腔形状的简化假设，在固定空腔体积下得出并最小化了几何学项。发现优化的腔体在大气泡体积上几乎是球形的，而在小体积下，腔具有镜头状的形状。优化的形状用于分析IMC的最低工作，事实证明，该功能明显小于均质气蚀的最低工作，因此表明在超声诱导的膜透气化过程中以及在外伤性脑损伤中，IMC在形成膜缺陷中的主要作用。