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(MSCs)是膜结合的蛋白质，它使水和溶质分子对膜变形做出反应而进出细胞(23)。通过让细胞对渗透变化做出反应，它们对生命是必不可少的。真核间充质干细胞参与感觉方式，如触摸、声音、液体平衡和血压(15，25)。这些通道的问题与心律失常、肌肉营养不良、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MSCs(大电导MSCL和小电导MSCs)是最好的理解。这可能有助于通过抗生素攻击细菌(29)，并可能有助于理解特征不佳的真核间充质干细胞(30)。然而，关于细菌间充质干细胞，特别是真核间充质干细胞的重要信息仍然缺失。对于细菌通道，MSCL通道已经以闭合形式结晶，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)，或者对必要的距离不敏感(35)。我们建议使用单分子荧光能量转移(SmFRET)来研究MSCL的开放和闭合状态，这是我帮助发明的一项技术(36，37)。这将得到分子动力学的补充，由Klaus Schulten(U.Illinois，Urbana-Champaign)(38，39)领导，他研究了MSCL/MSCs通道(1，3，4，6，7)。我们的另一位合作者Boris Martinac(澳大利亚Victor Chang研究所)广泛研究了MSCL/MSCs(32-35，40-46)。我们将研究MSCL是如何开放的。领先的候选模型是桶-杆模型，其中一个跨膜螺旋(TM1)移动，以及螺旋-倾斜模型(47)，两个螺旋(TM1和TM2)移动。(47)。我们在打开和关闭状态下都形成了MSCL通道，发现smFRET表明两个螺旋都发生了运动，导致了孔径2.8 nm的变化。因此，我们强烈支持螺旋倾斜模型。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(G22N)(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模拟来研究孔道打开的过程，我们已经成功地通过强迫水通过孔道来模拟孔洞的打开。最后，与纽约州立大学水牛城的Philip Gottlieb合作，我们在一个巨大的(&gt；1 MD)真核MSC上有了初步的结果，称为(四聚体)Piezo1(12，30，50)。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My Fiona技术(20))来研究选择的位置。结果表明，四聚体的N-N距离约为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.