Measuring the Opening of the Mechanosensitive Channel through smFRET & Molecular

描述（由申请人提供）：机械敏感离子通道（MSC）是膜结合蛋白质，可使水和溶质分子响应膜变形流入和流出细胞（23）。它们通过让细胞对渗透压变化做出反应而对生命至关重要（24）。真核MSC参与感觉方式，如触觉、声音、液体平衡和血压（15，25）。这些通道的问题与心律失常、肌营养不良、神经胶质瘤、病理性疼痛、神经前庭障碍和肿瘤转移有关（13，26 - 28）。来自细菌的MSC（大电导，MscL和小电导，MscS）是最好理解的。这可能有助于通过抗生素攻击细菌（29），并可能有助于了解真核MSC，其特征很差（30）。然而，关于细菌MSC，特别是真核MSC的重要信息缺失。对于细菌通道，MscL通道以封闭形式结晶，但不以开放形式结晶（31）。MscL的开孔已经通过几种集成技术进行了实验测试，包括EPR和集成FRET，但系统误差可能导致高估（32）或低估（33，34），或者对所需的距离不敏感（35）。我们建议使用单分子荧光能量转移（smFRET）研究MscL的开放和闭合状态，这是我帮助发明的一种技术（36，37）。这将补充分子动力学，由克劳斯舒尔滕（美国）。Illinois，Urbana-Champaign）（38，39）谁研究了MscL/MscS渠道（1，3，4，6，7）.我们的另一位合作者Boris Martinac（维克托张研究所，澳大利亚）广泛研究了MscL/MscS（32- 35，40 -46）。 我们将学习如何打开MscL。主要的候选模型是桶-板条模型，其中一个跨膜螺旋（TM 1）移动，以及螺旋-倾斜模型（47），其中两个螺旋（TM 1和TM 2）移动。（四十七）。我们已经形成了MscL通道的开放和关闭状态，并发现smFRET表明，两个螺旋移动，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，这是一个重大争议的来源（48，49）。我们有初步的smFRET数据，这表明CP结构域是解离的。此外，我们建议通过使用自发打开和关闭的突变体（G22 N）（49）研究通道的构象动力学来研究通道与其周围膜之间的相互作用。我们还建议通过独立的MD模拟研究通道开放的过程，我们已经成功地模拟了迫使水通过通道的孔隙的开放。 最后，与Philip Gottlieb，SUNY，布法罗合作，我们对一个巨大的（>1 MD）真核MSC，称为（四聚体）Piezo 1（12，30，50）有了初步结果。我们使用SimPull（17）来分离单个通道，并使用超分辨率荧光技术gSHRImP（18，51）和SHREC（19）（源自我的FIONA技术（20））来研究选择的位置。结果表明，四聚体的N-到-N距离为~52 nm。

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.