Measuring the Opening of the Mechanosensitive Channel through smFRET & Molecular

通过 smFRET 测量机械敏感通道的开口

• 批准号: 8760792
• 负责人: PAUL R SELVIN
• 金额: $ 29.04万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8760792
• 关键词: Antibiotics; Arrhythmia; Australia; Bacteria; Behavior; Binding Proteins; Biophysics; Blood Pressure; Buffaloes; Caliber; Cell Volumes; Cells; Collaborations; Complex; Cysteine; Cytoplasmic Tail; DNA Sequence Rearrangement; Data; Derivation procedure; Energy Transfer; Equilibrium; Erythrocytes; Escherichia coli; Fluid Balance; Fluorescence; Fluorescence Resonance Energy Transfer; Geometry; Glioma; Homology Modeling; Illinois; Institutes; Ion Channel; Label; Length; Life; Light; Lipids; Mammalian Cell; Measures; Membrane; Migraine; Modality; Modeling; Molecular; Molecular Conformation; Movement; Muscular Dystrophies; Mutation; Mycobacterium tuberculosis; Nail plate; Neoplasm Metastasis; Pain; Positioning Attribute; Potassium Channel; Probability; Process; Protein Conformation; Proteins; Relative (related person); Resolution; Sensory; Shapes; Simulate; Site; Source; Structure; Techniques; Testing; Time; Touch sensation; Validation; Water; analog; base; flexibility; human disease; millisecond; molecular dynamics; monomer; mutant; nanometer; patch clamp; prevent; public health relevance; research study; response; simulation; single molecule; single-molecule FRET; solute; sound; tumor; virtual; water flow

DESCRIPTION (provided by applicant): Mechanosensitive ion channels (MSCs) are membrane-bound proteins that let water and solute molecules flow in and out of the cell in response to membrane deformation (23). They are essential to life by letting the cell respond to osmotic changes (24). Eukaryotic MSCs are involved in sensory modalities, like touch, sound, fluid balance, and blood pressure (15,25). Problems with these channels are implicated in cardiac arrhythmia, muscular dystrophy, glioma, pathological pain, neurovestibular disturbances, and tumour metastasis (13,26- 28). MSCs (of large conductance, MscL, and of small conductance, MscS) from bacteria are best understood. This may aid in attacking bacteria via antibiotics (29) and may help understand the eukaryotic MSCs, which are poorly characterized (30). Yet vital information about the bacterial MSCs and especially eukaryotic MSCs, are missing. For the bacterial channels, the MscL channel has been crystallized in the closed form, but not in the open form (31). The open pore of the MscL has been experimentally tested via several ensemble techniques, including EPR and ensemble FRET, but systematic errors likely result in an overestimation (32), or an underestimation (33,34), or have not been sensitive to the requisite distances (35). We propose to study the open and closed states of MscL using single molecule fluorescence energy transfer (smFRET), a technique I helped to invent (36,37). This will be supplemented with molecular dynamics, led by Klaus Schulten (U. Illinois, Urbana-Champaign) (38,39) who has studied the MscL/MscS channels (1,3,4,6,7). Our other collaborator, Boris Martinac (Victor Chang Institute, Australia) has extensively studied the MscL/MscS (32-35,40-46). We will study how the MscL opens. The leading candidates are the barrel-stave model, with one transmembrane helices (TM1) moving, and the helix-tilt model (47), with two helices (TM1 and TM2) moving. (47). We have formed the MscL channel in both the open and closed state, and find that smFRET indicates that both helices move, leading to a change in the pore diameter of 2.8 nm. We therefore argue strongly for the helix-tilt model. We also propose to study the behavior of the cytoplasmic (CP) helix, a source of significant controversy (48,49). We have preliminary smFRET data, which argues that the CP domain is dissociated. Furthermore, we propose to investigate the interaction between the channel and its surrounding membrane by studying the conformational dynamics of the channel using a mutant (G22N) (49) which spontaneously opens and closes. We also propose to investigate the process of channel opening by independent MD simulations for which we have successfully simulated the opening of the pore by forcing water through the channel. Finally, in collaboration with Philip Gottlieb, SUNY, Buffalo, we have preliminary results on a huge (>1 MD) eukaryotic MSC, called (tetrameric) Piezo1 (12,30,50). We use both SimPull (17) to isolate a single channel, and super-resolution fluorescence techniques, gSHRImP (18,51) and SHREC (19) (derived from my FIONA technique (20)), to study select positions. Results suggest that the N-to-N distance of the tetramer is ~52 nm.

描述（由申请人提供）：机械敏感的离子通道（MSC）是膜结合的蛋白，可让水和溶质分子以膜变形而流入和流出细胞（23）。通过让细胞应对渗透变化，它们对生活至关重要（24）。真核MSC参与感觉方式，例如触摸，声音，流体平衡和血压（15,25）。这些通道的问题与心律不齐，肌肉营养不良症，神经胶质瘤，病理疼痛，神经粘卷菌障碍和肿瘤转移有关（13,26-28）。最好理解细菌的MSC（大型电导，MSCL和小电导，MSC）。这可能有助于通过抗生素攻击细菌（29），并有助于理解其特征不佳的真核MSC（30）。然而，缺少有关细菌MSC，尤其是真核MSC的重要信息。对于细菌通道，MSCL通道已以封闭形式结晶，但没有以开放形式结晶（31）。 MSCL的开放孔已通过包括EPR和集合品格在内的几种集合技术进行了实验测试，但是系统的错误可能导致高估（32）或低估（33,34），或者对必要距离不敏感（35）。我们建议使用单分子荧光能量转移（SMFRET）研究MSCL的开放状态和封闭状态，这是我帮助发明的一种技术（36,37）。这将补充由Klaus Schulten（U. Illinois，Urbana-Champaign）领导的分子动力学（38,39），他研究了MSCL/MSCS通道（1,3,4,6,7）。我们的其他合作者鲍里斯·马蒂纳克（Boris Martinac）（澳大利亚维克多·张研究所（Victor Chang Institute））已广泛研究了MSCL/MSC（32-35,40-46）。 我们将研究MSCL的打开方式。领先的候选者是桶形旋转模型，具有一个跨膜螺旋（TM1）移动，而螺旋倾斜模型（47），两个螺旋（TM1和TM2）移动。 （47）。我们已经在开放状态和封闭状态下形成了MSCL通道，发现SMFRET表示这两个螺旋都会移动，从而导致孔径的变化为2.8 nm。因此，我们强烈主张螺旋倾斜模型。我们还建议研究细胞质（CP）螺旋的行为，这是引起重大争议的根源（48,49）。我们有初步的SMFRET数据，该数据认为CP域是解离的。此外，我们建议使用突变体（G22N）（49）（49）（自发打开并关闭）来研究通道及其周围膜之间的相互作用。我们还建议通过独立的MD模拟来研究通道开放的过程，我们通过强迫水通过通道成功模拟了孔的开口。 最后，与菲利普·戈特利布（Philip Gottlieb），纽约州立大学（SUNY），布法罗（Buffalo）合作，我们在巨大（> 1 md）真核生物MSC上获得了初步结果，称为（四聚体）Piezo1（12,30,50）。我们同时使用Simpull（17）来分离单个通道和超分辨率荧光技术，GSHRIMP（18,51）和SHREC（19）（源自我的Fiona Technique（20）），以研究某些位置。结果表明，四聚体的n-to-N距离为〜52 nm。