Biophysics of Large Membrane Channels

大膜通道的生物物理学

基本信息

批准号：
10691786
负责人：
sergey bezrukov
金额：
$ 167.69万
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/10691786
关键词：
Agreement Anions Anthrax disease Architecture Bacillus anthracis Bacillus brevis Behavior Binding Biological Assay Biological Process Biophysics Calcium Cell Membrane Permeability Cells Characteristics Charge Chemical Engineering Clostridium botulinum Clostridium perfringens Communication Complex Crystallization Dependence Development Diffuse Diffusion Disease Electrical Resistance Electrochemistry Electrophysiology (science)Equilibrium Escherichia coli Event Fluorescence Free Energy Frequencies Geometry Hela Cells Hemolysin Hexokinase 2 In Vitro Inclusion Bodies Individual Interphase Cell Ion Channel Ions Ligation Measurement Measures Membrane Membrane Lipids Membrane Potentials Membrane Proteins Methods Mitochondria Modeling Molecular Motion Mycobacterium smegmatis Organism Outer Mitochondrial Membrane Parkinson Disease Pathology Peptides Peripheral Pharmacology Play Positioning Attribute Process Property Proteins Pseudomonas aeruginosa Radial Reaction Recombinant Proteins Regulation Reporting Research Personnel Resistance Resolution Respiration Role SECTM1 gene Shapes Side Spectrum Analysis Staphylococcus aureus Structure Study Section Sum Surface Time Toxic effect Toxin Transmembrane Transport Transport Process Tubulin VDAC1 gene Voltage-Dependent Anion Channel Water aerolysin alpha synuclein analog base beta barrel biophysical properties comparative density deviant dimer driving force experimental study gramicidin A interest mechanical properties mitochondrial metabolism molecular dynamics mutant nanopore new therapeutic target peptide drug protein complex rational design reconstitution recruit release of sequestered calcium ion into cytoplasm sensor simulation single molecule solute sugar synucleinopathy therapeutic target voltage voltage gated channel

项目摘要

I. Voltage-activated complexation of alpha-synuclein with beta-barrel channels and its inhibition as a potential therapeutic target for Parkinsons disease treatment Voltage-activated protein complexation is the process by which a transmembrane potential drives complex formation between a membrane-embedded channel and a soluble or membrane-peripheral target protein. Metabolite and calcium flux across the mitochondrial outer membrane was shown to be regulated by voltage-activated complexation of the voltage-dependent anion channel (VDAC) and either dimeric tubulin or alpha-synuclein (aSyn). However, the roles played by VDACs characteristic attributes its anion selectivity and voltage gating behavior have remained unclear. During this reporting period we have conducted a comparative analysis of in vitro measurements of voltage-activated complexation of aSyn with three well-characterized beta-barrel channels VDAC, MspA, and alpha-Hemolysin that differ widely in their organism of origin, structure, geometry, charge density distribution, and voltage gating behavior. The voltage dependences of the complexation dynamics for the different channels were observed to differ quantitatively but have similar qualitative features. In each case, energy landscape modeling describes the complexation dynamics in a manner consistent with the known properties of the individual channels, while voltage gating does not appear to play any role. The reaction free energy landscapes thus calculated reveal a common physical mechanism of complexation for all three channels, together with a non-trivial dependence of the complex stability on the surface density of aSyn. It is well-recognized that involvement of aSyn in Parkinsons disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently, we established that aSyn can regulate mitochondrial function by voltage-activated complexation with VDAC described above. When complexed with aSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites though the mitochondrial outer membrane. Further, aSyn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of aSyn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. This year we studied an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on aSyn membrane binding, and hence on aSyn complex formation with VDAC and translocation through it. In electrophysiology experiments, the addition of HK2p at micromolar concentrations to the same side of the membrane as aSyn results in a dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of aSyn from lipid membranes. Experiments with HeLa cells using proximity ligation assay confirmed that HK2p impedes aSyn entry into mitochondria. Our results demonstrate that it is possible to regulate aSynVDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate aSyn mitochondrial toxicity in PD and other synucleinopathies. II. The single residue K12 governs the exceptional voltage sensitivity of VDAC gating VDAC is the most abundant protein in the mitochondrial outer membrane (MOM) and is the primary conduit for ions and water-soluble metabolites such as ATP and ADP to cross the MOM. As such, VDAC plays a central role in the regulation of MOM permeability and mitochondrial metabolism, and in communication between mitochondria and the rest of the cell. VDAC responds to a transmembrane potential by gating, i.e. transitioning to one of a variety of low-conducting states of unknown structure. The gated state results in nearly complete suppression of multivalent mitochondrial metabolite (such as ATP and ADP) transport while enhancing calcium transport. Voltage gating is a common property of beta-barrel channels and has been observed in bacterial outer membrane porins as well as in anthrax, aerolysin, and alpha-Hemolysin toxins, but VDAC gating is anomalously sensitive to transmembrane potential. This reporting period we have shown that a single residue in the pore interior, K12, is responsible for most of VDACs voltage sensitivity. Using the analysis of over 40 microseconds of atomistic molecular dynamics (MD) simulations, we explored correlations between motions of charged residues inside the VDAC pore and geometric deformations of the beta-barrel. Residue K12 is bistable; its motions between two widely separated positions along the pore axis enhance the fluctuations of the beta-barrel and augment the likelihood of gating. Single channel electrophysiology of various K12 mutants reveals a dramatic reduction of the voltage-induced gating transitions. The crystal structure of the K12E mutant at a resolution of 2.6 indicates a similar architecture of the K12E mutant to the wild type; however, 60 microseconds of atomistic MD simulations using the K12E mutant showed restricted motion of residue 12, due to enhanced connectivity with neighboring residues, and diminished amplitude of barrel motions. We thus conclude that beta-barrel fluctuations, governed particularly by residue K12, drive VDAC gating transitions. III. Intrinsic diffusion resistance of a membrane channel, mean first-passage times between its ends, and equilibrium unidirectional fluxes Diffusion resistance is an important characteristic of channel-facilitated membrane transport that is widely used in chemical engineering, electrochemistry, and cell biophysics. It is a diffusion analog of the electrical resistance, relating the steady-state diffusive flux of solute molecules through a membrane channel with the driving force of the transport process, the solute concentration difference in the two reservoirs separated by the membrane. This reporting period we derived analytical expressions for the diffusion resistance in the case of a cylindrically symmetric blocker whose axis coincides with the axis of a cylindrical nanopore in two limiting cases where the blocker radius changes either smoothly or abruptly. Comparison of our theoretical predictions with the results obtained from Brownian dynamics simulations shows good agreement between the two. We have also established a general relation between the channel diffusion resistance and the mean first-passage times of the solute molecules between the channel openings. Specifically, we have shown that this direction-independent characteristic of transport is equal to the sum of the direction-dependent mean first-passage times, divided by the molecule partition function in the channel. Our analysis is based on the consideration of the equilibrium unidirectional fluxes flowing through the channel in opposite directions. The approach is quite general in the sense that it does not appeal to any specific model of the channel and, therefore, is universally applicable to transport in channels of arbitrary shape and tortuosity, at arbitrary interaction strength of solute molecules with the channel walls. This result promises to be of great value in computing the intrinsic diffusion resistance of the channel numerically, as it allows researchers to avoid dealing with multiple problems in analyzing transport under non-equilibrium conditions.

I.α-核蛋白与β-桶通道的电压激活络合及其抑制作用是帕金森氏病治疗的潜在治疗靶点电压激活的蛋白质络合是跨膜电势驱动膜上包裹的通道与可溶性或膜 - 外膜靶蛋白之间的复合形成的过程。线粒体外膜的代谢产物和钙通量显示受电压依赖性阴离子通道（VDAC）的电压激活络合（VDAC）和二聚体微管蛋白或α-突触核蛋白（Asyn）的调节。但是，VDACS特征赋予其阴离子的选择性和电压门控行为的角色尚不清楚。在此报告期间，我们对ASYN与三个特征良好的β-桶通道VDAC，MSPA和Alpha-Hemolysin的电压激活络合的体外测量进行了比较分析。观察到不同通道的络合动力学的电压依赖性在定量上有所不同，但具有相似的定性特征。在每种情况下，能量景观建模都以与各个通道的已知特性一致的方式描述络合动力学，而电压门似乎没有任何作用。因此，计算出的反应自由能景观揭示了所有三个通道的共同物理机制，以及复杂稳定性对ASYN表面密度的非平凡依赖性。公认的是，ASYN参与帕金森氏病（PD）是复杂的，难以在细胞和分子水平上追踪。最近，我们确定ASYN可以通过与上述VDAC进行电压激活的络合来调节线粒体功能。当与ASYN复合时，VDAC孔被部分阻塞，从线粒体外膜减少ATP/ADP和其他代谢产物的运输。此外，Asyn可以通过VDAC转移到线粒体中，在那里它会干扰线粒体呼吸。将ASYN募集到含VDAC的脂质膜上似乎是阻塞和易位过程的关键先决条件。今年，我们研究了HK2P的抑制作用，HK2P是来自己糖激酶2的线粒体靶向N端的小膜结合肽，对ASYN膜结合，因此与Asyn复合物形成了与VDAC的Asyn复合物形成，并通过其易位。在电生理实验中，随着ASYN的同一浓度，在微摩尔浓度的同一侧将HK2P添加，因为ASYN会以浓度依赖性的方式显着减少阻塞事件的频率，并报告了络合性抑制作用。使用两种互补方法测量蛋白质 - 膜结合，双层甲板分析和荧光相关光谱法，我们发现HK2P诱导ASYN脱离脂质膜。使用接近连接测定的HeLa细胞实验证实，HK2P阻碍了ASYN进入线粒体。我们的结果表明，可以通过理性设计的肽来调节AsynvDAC络合，从而在寻找肽疗法时提出新的途径，以减轻ASYN线粒体在PD和其他突触中的毒性。 ii。单个残基K12控制VDAC门控的特殊电压灵敏度 VDAC是线粒体外膜（MOM）中最丰富的蛋白质，是离子和水溶性代谢物（例如ATP和ADP）越过妈妈的主要管道。因此，VDAC在调节MOM渗透性和线粒体代谢以及线粒体与细胞其余部分之间的通信中起着核心作用。 VDAC通过门控对跨膜电位做出反应，即过渡到未知结构的各种低传导状态之一。封闭状态几乎完全抑制了多价线粒体代谢产物（例如ATP和ADP）的转运，同时增强了钙转运。电压门控是β-桶通道的一种共同特性，在细菌外膜孔蛋白以及炭疽，气动蛋白和α-羟蛋白蛋白毒素中都观察到，但VDAC门控对跨膜电位异常敏感。在此报告期间，我们已经表明，孔内部中的单个残基K12负责大多数VDAC电压敏感性。利用对40微秒的原子分子动力学（MD）模拟的分析，我们探索了VDAC孔内带电残基的运动与β-桶的几何变形之间的相关性。残留K12是可动的；它在沿孔轴的两个广泛分离位置之间的运动增强了β桶的波动，并增加了门控的可能性。各种K12突变体的单个通道电生理学揭示了电压诱导的门控跃迁的显着降低。 K12E突变体的晶体结构在2.6分辨率下表明K12E突变体的结构与野生型相似。然而，由于与邻近残基的连通性增强以及枪管运动振幅的减小，使用K12E突变体的60微秒原子MD模拟显示了残基12的限制。因此，我们得出的结论是，β-桶波动特别受残基K12的控制，驱动VDAC门控转变。 iii。膜通道的固有扩散性，其末端之间的平均第一步时间和平衡单向通量扩散耐药性是通道传播膜转运的重要特征，该特征广泛用于化学工程，电化学和细胞生物物理学。它是电阻的扩散类似物，将溶质分子的稳态扩散通量通过膜通道与传输过程的驱动力相关联，这是由膜分离的两个储层的溶质浓度差。在此报告期间，我们在圆柱对称阻滞剂的情况下得出了扩散抗性的分析表达式，其轴与圆柱纳米孔的轴相吻合，在两个限制器半径平稳变化或突然变化的情况下。我们的理论预测与从布朗动力学模拟获得的结果的比较表明两者之间的良好一致性。我们还建立了通道扩散抗性与通道开口之间溶质分子的平均第一通道时间之间的一般关系。具体而言，我们已经表明，这种与方向无关的传输特征等于方向依赖的平均第一邮箱时间的总和，除以通道中的分子分区函数。我们的分析是基于考虑沿相反方向流过通道的平衡单向通量的考虑。从某种意义上说，这种方法是相当普遍的，它不吸引任何特定模型的通道模型，因此，在溶质分子与通道壁的任意相互作用强度下，普遍适用于任意形状和曲折通道的运输。该结果有望在数值上计算通道的固有扩散阻力方面具有很大的价值，因为它使研究人员能够避免在非平衡条件下分析传输时处理多个问题。