Biophysics of Large Membrane Channels

大膜通道的生物物理学

基本信息

批准号：
7968454
负责人：
sergey bezrukov
金额：
$ 74.47万
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/7968454
关键词：
Achievement Adamantane Address Adenine Nucleotide Translocase Affect Alamethicin Anesthetics Anthrax disease Apoptosis Area Behavior Binding Biochemical Reaction Biological Biophysics C-terminal Cell Membrane Permeability Cells Charge Complex Crowding Crystallography Cyclodextrins Cytoplasm Cytoskeletal Proteins Cytoskeleton Cytosol DNA-Protein Interaction Development Diffusion Disease Drug Delivery Systems Drug Design Educational process of instructing Electrostatics Entropy Environment Equilibrium Escherichia coli Eukaryota Glycolysis Goals Gramicidin Halothane Hemolysin Homeostasis Hydration status Ion Channel Kinetics Laboratories Length Life Ligands Link Lipid Bilayers Lipids Liquid substance Measures Membrane Membrane Potentials Microtubules Mitochondria Modeling Molecular Molecular Probes Organelles Organism Outer Mitochondrial Membrane Oxidative Phosphorylation Paclitaxel Peptides Pharmaceutical Preparations Phospholipids Physiological Play Proteins Pseudomonas aeruginosa Pseudomonas syringae Reaction Regulation Research Resolution Respiration Role Salts Side Site Sodium Chloride Solutions Staphylococcus aureus Stress Structure Surface Tail Time Toxin Transmembrane Transport Trichoderma Tubulin Tubulin Interaction Variant Vincristine Voltage-Dependent Anion Channel Warburg Effect Water Work anthrax protective factor aqueous base cancer cell clinically relevant gramicidin A molecular recognition nanopore novel strategies particle porin protein folding receptor reconstitution research study sensor simulation single molecule solute sugar syringomycin E theories voltage

项目摘要

Large ion channels are recognized as both the gateways of metabolite exchange and multifunctional membrane receptors. They are also critical components of many toxins. To study channels under precisely controlled conditions, we purify the channel-forming proteins produced by different organisms and then reconstitute them into planar lipid bilayer membranes. We explore a number of channel-forming proteins which include mammalian VDAC (Voltage-Dependent Anionic Channel from the outer membrane of mitochondria), Anthrax Protective Antigen (from Bacillus anthracis), OmpF (general bacterial porin from Escherichia coli), LamB (sugar-specific bacterial porin from Escherichia coli), alpha-Hemolysin (toxin from Staphylococcus aureus), OprF (porin from Pseudomonas aeruginosa), Alamethicin (amphiphilic peptide toxin from Trichoderma viride), and Syringomycin E (lipopeptide toxin from Pseudomonas syringae). We think that combining work with this broad variety of channels of different origin, structure, and function in one laboratory is the most effective strategy towards elucidation of major physical mechanisms of their regulation. I. Physical theory of channel-facilitated metabolite transport. Our effort in physical theory concentrates on further development of the continuum diffusion model of solute dynamics in a membrane channel. The most important advance of this year was to apply our analytical approach to the so-called entropy potentials and to show the importance of particle-particle interactions in breaking the particle flux symmetry. Water-filled pores of biological channels usually have complex geometry that only rarely can be approximated by a cylinder. For example, high-resolution crystallography of bacterial porins and other large channels demonstrates that their pores can be envisaged as tunnels whose cross-sections change significantly along the channel axis. For some of them, variation in cross-section area exceeds an order of magnitude, which leads to the so-called entropic wells and barriers in theoretical description of transport through such structures. To approach this complex problem, we analyzed transport through conical channels that is driven by the difference in particle concentrations on the two sides of the membrane. Indeed, fluxes of non-interacting particles through the same channel, inserted into the membrane in two opposite orientations, are equal because of the detailed balance. We have shown that this flux symmetry is broken by particle-particle interactions, so that one of the orientations the orientation corresponding to the increasing channel cross section in the direction of transport can be much more efficient under the same external conditions. Our analytical results were confirmed using three-dimensional Brownian dynamics simulations. II. Physical interactions in transport regulation. This year we were able to address a wide scope of questions ranging from van der Waals interactions of particles in liquids to electrostatic effects in channel selectivity and conductance. One of our achievements was the elucidation of the lipid-packing-dependent partitioning of the prototypical anesthetic halothane into lipid bilayers. Using gramicidin A channel as a molecular probe, we found that its sensitivity to the anesthetic is highly lipid dependent. Specifically, exposure of membranes made of lamellar DOPC to halothane in concentrations close to clinically relevant reduces channel life-times by an order of magnitude. At the same time, gramicidin channels in membranes of non-lamellar DOPE are little, if at all, affected by halothane. We attribute this difference in channel behavior to a difference in the stress of lipid packing into a planar lipid bilayer, wherein the higher stress of DOPE packing reduces halothane partitioning into the hydrophobic interior. Thus, our findings suggest a new role for the physical interactions originating from the stress of lipid packing, revealing a previously unknown mechanism of anesthetic efficacy regulation. Another achievement was to use an ion channel nanopore as a single-molecule sensor to follow the effect of different salts on the adamantane-cyclodextrin complexation reaction. Since the pioneering experiments of Hofmeister on the salting out of aqueous solutions of proteins, a vast body of research has taught us that cosolutes can increase or decrease complex stability, whether preferentially excluded from or attracted to the surfaces of the associating molecules. Now studies of the underlying kinetics at a single-molecule level open the possibility of instructive inquiry into the molecular basis of the phenomenon. Surprisingly, we found that not only the stability of the complex, as measured by the life time of the inclusion complex, but also the accessibility of the complexation site can be critically increased by preferentially excluded cosolutes. Significance of these findings follows from the fact that all biochemical reactions, such as protein folding, drug binding to the target, protein-ligand and protein-DNA interactions, require release or restructuring of water layers associated with interacting molecular surfaces in the crowded environment of the cell. Therefore, understanding the mechanisms of molecular surface hydration is crucially important in resolving the fundamental questions of molecular recognition and assembly, drug design, and transport regulation. III. A new role for the old protein: Tubulin in regulation of mitochondria respiration. Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation involving the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. A long-standing puzzle is that in permeabilized cells, adenine nucleotide translocase (ANT) is less accessible to cytosolic ADP than in isolated mitochondria. We solve this puzzle by finding a missing player in the regulation of MOM permeability: the cytoskeletal protein tubulin. We show that nanomolar concentrations of dimeric tubulin induce voltage-sensitive reversible closure of VDAC reconstituted into planar phospholipid membranes. Tubulin strikingly increases VDAC voltage sensitivity and at physiological salt conditions could induce VDAC closure at <10 mV transmembrane potentials. Experiments with isolated mitochondria confirm these findings. Tubulin added to isolated mitochondria decreases ADP availability to ANT, partially restoring the low MOM permeability (high apparent Km for ADP) found in permeabilized cells. Our findings suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC and tubulin at the mitochondria-cytosol interface. This tubulin-VDAC interaction requires tubulin anionic C-terminal tail peptides. The significance of this interaction may be reflected in the evolutionary conservation of length and anionic charge in the C-terminal tails throughout eukaryotes, despite wide changes in the exact sequence. Additionally, tubulins that have lost significant length or anionic character of C-terminal tails are only found in cells that do not have mitochondria. To conclude, the discovered VDAC/tubulin interaction provides a previously absent link between Ca2+ homeostasis, cytoskeleton/microtubule activity, mitochondria, and apoptosis. This may help to identify the mechanisms of mitochondria-associated action of chemotherapeutic microtubule-targeting drugs such as paclitaxel and vincristine and also to understand why and how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (Warburg effect).

大离子通道被认为是代谢产物交换和多功能膜受体的门户。它们也是许多毒素的关键组成部分。为了在精确控制条件下研究通道，我们净化了由不同生物体产生的通道形成蛋白，然后将它们重构为平面脂质双层膜。 We explore a number of channel-forming proteins which include mammalian VDAC (Voltage-Dependent Anionic Channel from the outer membrane of mitochondria), Anthrax Protective Antigen (from Bacillus anthracis), OmpF (general bacterial porin from Escherichia coli), LamB (sugar-specific bacterial porin from Escherichia coli), alpha-Hemolysin （来自金黄色葡萄球菌的毒素），OPRF（铜绿假单胞菌的porin），阿拉米糖蛋白（来自Trichoderma viride的两亲性肽毒素）和肌霉素E（来自假单胞菌的脂肽毒素）。我们认为，将工作与一个实验室中不同起源，结构和功能的各种渠道相结合是阐明其调节主要物理机制的最有效策略。 I.通道促进代谢物转运的物理理论。我们在物理理论方面的努力集中于在膜通道中进一步开发溶质动力学的连续扩散模型。今年最重要的进步是将我们的分析方法应用于所谓的熵电位，并显示颗粒粒子相互作用在破坏粒子通量对称性中的重要性。生物通道的水孔通常具有复杂的几何形状，而圆柱体很少能近似。例如，细菌孔蛋白和其他大通道的高分辨率晶体学表明，可以将它们的毛孔作为隧道的设想，其横截面沿通道轴很大变化。对于其中的某些人来说，横截面区域的变化超过了一个数量级，这导致了通过此类结构对传输的理论描述中所谓的熵井和障碍。为了解决这个复杂的问题，我们通过圆锥形通道分析了由膜上两侧颗粒浓度差异驱动的传输。实际上，由于详细的平衡，插入两个相反方向的膜的非相互作用颗粒的通量是相等的。我们已经表明，这种通量对称性被粒子粒子相互作用打破，因此在相同的外部条件下，在运输方向上与增加通道横截面相对应的方向之一可以更有效。使用三维的布朗动力学模拟证实了我们的分析结果。 ii。运输调节中的物理相互作用。今年，我们能够解决从液体中颗粒的范围相互作用到通道选择性和电导中的静电效应的广泛问题。我们的成就之一是阐明了将原型麻醉晕螺旋脂质包装依赖性分配到脂质双层中。使用谷霉素A通道作为分子探针，我们发现其对麻醉的敏感性高度依赖于脂质。具体而言，层状DOPC制成的膜以接近临床相关的浓度暴露于阳oth，从而减少了通道的生命时间。同时，非固有涂料膜中的谷霉素通道很少，如果有的话，受硫烷的影响很小。我们将这种通道行为的差异归因于脂质填料应力的差异，将脂质双层的含量归因于平面脂质双层，其中较高的涂料堆积应力减少了硫烷的分配到疏水性内部。因此，我们的发现表明了源自脂质堆积的应力的物理相互作用的新作用，从而揭示了先前未知的麻醉疗效调节机制。另一个成就是使用离子通道纳米孔作为单分子传感器，以遵循不同盐对阿甘坦烷基旋克力化蛋白络合反应的影响。由于霍夫梅斯特（Hofmeister）从蛋白质水溶液中进行盐溶液的开创性实验，因此大量的研究告诉我们，无论是优先排除或吸引了相关分子的表面，宇宙上可以提高或降低复杂的稳定性。现在，对单分子水平的潜在动力学的研究开启了对现象的分子基础的指导性询问的可能性。令人惊讶的是，我们发现，不仅通过包容络合物的寿命来衡量的络合物的稳定性，而且综合位点的可及性也可以通过优先排除的颜色来批判性地增加。这些发现的意义来自以下事实：所有生化反应，例如蛋白质折叠，与靶标的药物结合，蛋白质 - 配体和蛋白质-DNA相互作用，都需要释放或重组与细胞拥挤环境中相互作用的分子表面相关的水层。因此，了解分子表面水合的机制对于解决分子识别和组装，药物设计和转运调控的基本问题至关重要。 iii。旧蛋白质的新作用：微管蛋白在线粒体呼吸调节中的作用。线粒体外膜（MOM）渗透性的调节具有双重重要性：在正常的代谢物和线粒体和细胞质之间的能量交换中，从而控制了呼吸，以及通过将凋亡因子释放到细胞质中而凋亡。然而，涉及莫姆的主要通道（VDAC）的调节机理仍然有争议。长期存在的难题是，在透化细胞中，腺苷核苷酸易位酶（ANT）比孤立的线粒体易于接近胞质ADP。我们通过在MOM渗透性的调节中找到缺失的玩家来解决这个难题：细胞骨架蛋白微管蛋白。我们表明，二聚体小管蛋白的纳摩尔浓度诱导了对电压的可逆闭合VDAC的可逆闭合，重构为平面磷脂膜。微管蛋白显着提高VDAC电压敏感性，在生理盐条件下，可以在<10 mV的跨膜电位下诱导VDAC闭合。分离的线粒体实验证实了这些发现。添加到分离的线粒体中添加的微管蛋白可降低ADP的可用性，部分恢复了透化细胞中发现的低MOM渗透性（ADP的高明显km）。我们的发现表明，先前未知的线粒体能量调节机理，该机制由线粒体 - 蛋白质界面的VDAC和小管蛋白控制。这种微管蛋白-VDAC相互作用需要微管蛋白阴离子C末端尾肽。尽管精确的序列发生了很大变化，但这种相互作用的意义可能反映在整个真核生物的C末端尾巴的长度和阴离子电荷的进化保护中。另外，仅在没有线粒体的细胞中发现了C末端尾巴的显着长度或阴离子特征的小管蛋白。总而言之，发现的VDAC/微管蛋白相互作用在Ca2+稳态，细胞骨架/微管活性，线粒体和凋亡之间提供了以前不存在的联系。这可能有助于确定化学治疗微管靶向药物（如紫杉醇和vincristine）与线粒体相关作用的机制，也了解为什么癌细胞优先使用效率低下的糖酵解而不是氧化磷酸化（WARBURG效应）。