Biophysics of Large Membrane Channels

大膜通道的生物物理学

基本信息

批准号：
10007484
负责人：
sergey bezrukov
金额：
$ 148.12万
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/10007484
关键词：
Accounting Address Affect Alamethicin Amino Acids Anesthetics Anions Architecture Bacillus anthracis Bacillus brevis Bacterial Toxins Binding Binding Proteins Biological Assay Biological Models Biophysics Biopolymers Bypass C-terminal Cations Cell physiology Cells Charge Classification Clinical Clostridium botulinum Clostridium perfringens Clostridium perfringens epsilon toxin Communication Complex Confocal Microscopy DNA DNA sequencing Data Set Dependence Detection Development Diffuse Diffusion Disease Dissociation Electrostatics Environment Equilibrium Escherichia coli Exhibits Fluorescence Goals Grain Hemolysin Hydrophobicity Individual Ion Channel Ions Learning Lipid Bilayers Lipids Liposomes Measures Membrane Membrane Lipids Membrane Potentials Membrane Proteins Methods Mitochondria Modeling Molecular Monitor Motivation Mycobacterium smegmatis N-terminal Nature Nutrient Organelles Organism Outer Mitochondrial Membrane Parkinson Disease Pathologic Pathology Pattern Peptides Peripheral Pharmaceutical Preparations Plants Play Polymers Process Protein translocation Proteins Pseudomonas aeruginosa Pseudomonas syringae Regulation Reporting Role Site Sodium Chloride Spectrum Analysis Staphylococcus aureus Surface System Techniques Technology Time Toxin Trichoderma Tubulin VDAC1 gene Voltage-Dependent Anion Channel Work alpha synuclein amphiphilicity base beta barrel copolymer density design deviant di-block copolymer experimental study gramicidin A grasp interest mechanical properties nanomolar nanopore neurophysiology new technology novel strategies physical property polypeptide protein folding real time monitoring reconstitution scaffold sensor sensory mechanism single molecule small molecule solute success sugar theories voltage

项目摘要

I. Membrane association of alpha-synuclein domains studied using VDAC nanopore reveals an unexpected binding pattern The exceptional diversity of interactions between peripheral membrane proteins and bilayer lipid membranes makes the membrane surface a rich scene for performing and regulating cellular functions. This same diversity presents significant experimental barriers to studies of interaction mechanisms among the components. One of the main challenges is that the energies of interaction between individual protein residues and lipid molecules are small; thus, statistical effects play a significant role. A variety of techniques to characterize binding of peripheral membrane proteins to liposome or planar lipid bilayer platforms have been developed, but it is increasingly clear that, except in the simplest of systems, no isolated technique yields a clear picture of the membrane binding process. In part, this is because these assays measure the average binding parameters of an ensemble of molecules, highlighting the need to characterize membrane-bound proteins at the single-molecule level. This year, we used the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane as a single-molecule probe for alpha-synuclein (a-syn), a protein of considerable clinical interest due to its well-established involvement in the pathology of Parkinson disease. It is well established that a-syn binding from solution to the surface of membranes composed of negatively charged and/or non-lamellar lipids can be characterized by equilibrium dissociation constants of tens of micromolar. However, we previously found that VDAC, reconstituted into planar bilayers of a plant-derived lipid, responds to a-syn at nanomolar solution concentrations. Now, using lipid mixtures that mimic the composition of mitochondrial outer membranes, we showed that functionally important binding does indeed take place in the nanomolar range. We demonstrated that the voltage-dependent rate at which a membrane-embedded VDAC nanopore captures a-syn is a strong function of membrane composition. Comparison of the nanopore results with those obtained by the bilayer overtone analysis of membrane binding demonstrated a pronounced correlation between the two datasets. The stronger the binding, the larger the on-rate, but with some notable exceptions. This leads to a tentative model of a-syn-membrane interactions, which assigns different lipid-dependent roles to the N- and C-terminal domains of a-syn accounting for both electrostatic and hydrophobic effects. As a result, the rate of a-syn capture by the nanopore is not simply proportional to the a-syn concentration on the membrane surface but found to be sensitive to the specific interactions of each domain with the membrane and nanopore. II. Real-time nanopore-based recognition of protein translocation success A growing number of new technologies are supported by a single- or multi-nanopore architecture for capture, sensing, and delivery of polymeric biomolecules. Nanopore-based single-molecule DNA sequencing is the premier example. This method relies on the uniform linear charge density of DNA, so that each DNA strand is overwhelmingly likely to pass through the nanopore and across the separating membrane. For disordered peptides, folded proteins, or block copolymers with heterogeneous charge densities, by contrast, translocation is not assured, and additional strategies to monitor the progress of the polymer molecule through a nanopore are required. This year we studied the translocation of a heterogeneously charged polypeptide using model-free detection of the effect of the polymer charge on the electrical environment inside the nanopore to monitor the translocation process of single polypeptides in real time. This was accomplished using selectivity tags regions of different but uniform charge density at the ends of a polypeptide that produce different selectivity of the nanopore to cations and anions, and hence ionic current levels, in a voltage-biased nanopore under a salt concentration gradient. We employed the natural, disordered diblock copolymer-like 140-amino-acid polypeptide alpha-synuclein, which comprises two such tags, a highly negatively charged C-terminal region (CT; 43 amino acids, total charge 15e) and a largely neutral N-terminal region (NT; 97 amino acids, total charge +3e). By using these features, we demonstrated a single-molecule method for direct, model-free, real-time monitoring of the translocation of a disordered, heterogeneously charged polypeptide through a nanopore. The two selectivity tags at the ends of the polypeptide enabled us to discriminate between a-syn translocation and retraction. Our results demonstrated exquisite sensitivity of polypeptide translocation to the applied transmembrane potential and proved the principle that nanopore selectivity reports on biopolymer substructure. We anticipate that the selectivity tag technique will be broadly applicable to nanopore-based protein detection, analysis, and separation technologies, and to the elucidation of protein translocation processes in normal cellular function and in disease. III. Mapping intra-channel diffusive dynamics of interacting molecules onto a two-site model: Crossover in flux concentration dependence Transport of various solutes through membrane channels is an extremely complex phenomenon. One of the reasons for this complexity is the interactions of solute molecules between themselves and with the channel. This year we addressed this problem by analyzing how these interactions affect the flux dependence on the solute concentration. The study focused on narrow membrane channels, where it was assumed that the molecules cannot bypass each other because of their hard-core repulsion. In addition, other short- and long-range solute-solute interactions were included into consideration. Such interactions make it impossible to develop an analytical theory for the flux in the framework of the continuum diffusion model of solute dynamics in the channel developed in our lab during recent years. To overcome this difficulty, we coarse-grained the diffusion model by mapping it onto a two-site one where the rate constants describing the solute dynamics were expressed in terms of the parameters of the initial diffusion model. This allowed us (i) to find an analytical solution for the flux as a function of the solute concentration and (ii) to characterize the solute-solute interactions by two dimensionless parameters. Such a characterization proved to be very informative as it resulted in a clear classification of the effects of the solute-solute interactions on the concentration dependence of the flux. Unexpectedly, it turned out that this dependence can be nonmonotonic, exhibiting a sharp maximum as a function of system parameters. In other words, we have found that the effect is quite nontrivial: The flux can reach a well-pronounced maximum at a certain optimal concentration, the value of which is defined by the interaction parameters. We hypothesize that this phenomenon may be used by nature as a sensory mechanism of a regulatory circuit, wherein an optimal solute concentration is reported upon by maximizing the transmembrane flux of the molecules.

I.使用VDAC纳米孔研究的α-突触核蛋白结构域的膜关联显示出意外的结合模式外周膜蛋白和双层脂质膜之间相互作用的异常多样性使膜表面成为执行和调节细胞功能的丰富场景。同样的多样性为组件之间相互作用机制的研究带来了重大的实验障碍。主要挑战之一是，单个蛋白质残基和脂质分子之间相互作用的能量很小。因此，统计效应起着重要作用。已经开发了各种表征周围膜蛋白与脂质体或平面脂质双层平台的结合的技术，但是越来越明显的是，除了最简单的系统中，没有隔离技术可以清楚地了解膜结合过程。部分是因为这些测定法测量了分子集合的平均结合参数，这突出了在单分子水平上表征膜结合蛋白的需求。今年，我们将线粒体外膜的电压依赖性阴离子通道（VDAC）用作单分子探针的单分子探针（A-Synn），这是一种相当大的临床兴趣的蛋白质，这是由于其良好的临床感兴趣蛋白质在帕克森病的病理学中涉及。可以很好地确定，从溶液到由负电荷和/或非层状脂质组成的膜表面的A-syn结合可以通过数十个微摩尔的平衡分离常数来表征。但是，我们以前发现，VDAC重组为植物衍生脂质的平面双层，在纳摩尔溶液浓度下对A-Syn响应。现在，使用模仿线粒体外膜组成的脂质混合物，我们表明确实在功能上重要的结合确实发生在纳摩尔范围内。我们证明了膜上包含的VDAC纳米孔捕获A-Syn的电压依赖性速率是膜组成的强大功能。纳米孔结果与膜结合的双层泛音分析获得的结果比较表明两个数据集之间有明显的相关性。绑定越强，率越大，但有一些显着的例外。这导致了A-syn-膜相互作用的暂定模型，该模型将不同的脂质依赖性作用分配给A-Syn的N-和C末端结构域，这构成了静电和疏水性效应。结果，纳米孔的A-syn捕获速率不仅与膜表面上的A-SYN浓度成正比，而且发现对每个结构域与膜和纳米孔的特定相互作用敏感。 ii。基于纳米孔的实时识别蛋白质易位成功越来越多的新技术由单纳波尔架架构支持，用于捕获，感应和传递聚合物生物分子。基于纳米孔的单分子DNA测序是首要示例。该方法依赖于DNA的均匀线性电荷密度，因此每个DNA链都有绝大多数通过纳米孔和分离的膜穿过。相比之下，对于无序的肽，折叠蛋白或具有异质电荷密度的共聚物，不保证易位，并且需要通过纳米孔监测聚合物分子的进度的其他策略。今年，我们使用模型无需检测到聚合物电荷对纳米孔内电气环境的影响，以实时监测单多肽的易位过程。这是使用在多肽末端具有不同但均匀电荷密度的选择性标签区域来完成的，该区域在盐浓度梯度下在电压偏置的纳米孔中产生纳米孔对阳离子和阴离子的选择性不同。我们采用了天然的，无序的二嵌段共聚物样140-氨基酸多肽α-核蛋白，其中包括两个这样的标签，一个高度负电荷的C端区域（CT; 43个氨基酸，43个氨基酸，总电荷15E）和很大的中性NT末端区域（NT; 97 Amino Amino Arino Acider; 97 Amino Acid cyleds; 97 Amino Acids，requal +3ee）， +3ee +3e。通过使用这些功能，我们展示了一种单分子方法，用于直接，无模型，实时监测通过纳米孔的无序，异质性多肽的易位。多肽末端的两个选择性标签使我们能够区分A-Syn易位和回缩。我们的结果表明，多肽转移到应用的跨膜电位上的精致敏感性，并证明了纳米孔的选择性报告生物聚合物亚结构的原则。我们预计，选择性标签技术将广泛适用于基于纳米孔的蛋白质检测，分析和分离技术，以及在正常细胞功能和疾病中阐明蛋白质易位过程。 iii。将相互作用分子的通道内扩散动力学映射到两个站点模型：磁通浓度依赖性的交叉各种溶质通过膜通道的运输是一种极其复杂的现象。这种复杂性的原因之一是溶质分子之间与信道之间的相互作用。今年，我们通过分析这些相互作用如何影响对溶质浓度的通量依赖性来解决这个问题。该研究集中在狭窄的膜通道上，假定该分子由于硬核排斥而无法绕过彼此。此外，考虑了其他短期和远程溶质 - 溶质相互作用。这种相互作用使得在近年来在我们实验室中开发的通道中溶质动力学的连续扩散模型的框架中，不可能为通量的分析理论开发出一种分析理论。为了克服这一难度，我们通过将扩散模型映射到两个站点的扩散模型中，其中描述溶质动力学的速率常数是根据初始扩散模型的参数表达的。这使我们（i）能够找到通量的分析解决方案，作为溶质浓度的函数，（ii）通过二小无参数来表征溶质 - 溶液相互作用。这种表征被证明是非常有用的，因为它导致了溶质 - 糖相互作用对通量浓度依赖性的影响的明确分类。出乎意料的是，事实证明，这种依赖性可能是非单调的，它表现出最大值作为系统参数的函数。换句话说，我们发现效果非常不平凡：通量可以在一定的最佳浓度下达到良好的最大值，其值是由相互作用参数定义的。我们假设这种现象可以被自然界用作调节回路的感官机制，其中通过最大化分子的跨膜通量来报告最佳的溶质浓度。