RII Track-4:NSF: Unravelling Gating Mechanisms of Ion Channels Using Computational and Experimental Ultrafast Vibrational Spectroscopy

钾离子通道是促进钾离子跨细胞膜运输的膜蛋白。它们在神经和肌肉放松、认知和调节血压方面发挥着关键作用。虽然K+通道已被广泛研究多年，但离子通道的激活机制仍然是一个持续争论的话题，无法用既定的实验方法解决。二维红外光谱（2D IR）是一种新兴的分析技术，具有特定于化学键的空间和高时间分辨率来探测蛋白质的结构变化。最近的技术进步允许将二维红外光谱应用于生理现实条件下的离子通道。本课题将分子动力学模拟与计算光谱学相结合，研究K+通道的活化机理。模拟将通过二维红外实验进行验证，并反过来用于设计新的实验，以提供对K+通道激活机制的最清晰见解。提出的工作将推导出K+通道函数的原子级描述。了解K+通道功能的机制是确定心律失常和癫痫等严重疾病病因的关键因素。这为离子通道药理学治疗方法的设计提供了前景。这个研究基础设施改进轨道4 EPSCoR研究人员（RII轨道4）项目将为特拉华大学（UD）的一位助理教授提供奖学金。电压门控K+通道（Kv）是一种完整的膜蛋白，可以根据电化学梯度选择性地传导K+离子穿过细胞膜。Kv的电压感应域（VSD）感知膜电位的变化并触发构象变化，从而导致通道的打开和关闭。尽管有丰富的核磁共振和x射线晶体学数据，但关于VSD功能的许多基本问题仍未解决。假设VSD的S4螺旋段在电压门控期间发生构象和/或水合变化，但这一假设从未通过直接结构测量来验证，因为大多数生物物理结构技术不能在外加电压下进行。二维红外光谱可以在特定的位置分辨率和生理条件下探测蛋白质结构，包括施加电压。它还可以探测在毫秒时间尺度上发生的VSD的门控动力学。近年来，二维红外光谱的灵敏度显著提高，使其特别适合研究离子通道。这项工作将结合分子动力学模拟和计算二维红外光谱来阐明电压激活过程中VSD的构象和水合变化。现有的VSD结构模型将通过二维红外实验进行模拟和测试。在与实验比较的基础上，对模型进行改进或开发新的模型。模拟也将用于设计新的二维红外实验。我们的目标是确定VSD的相关构象以及它们在电压激活期间发生的顺序。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

K+ channels are membrane proteins that facilitate the transport of K+ ions across the cell membranes. They play key roles in nerve and muscle relaxation, cognition, and regulation of blood pressure. While K+ channels have been extensively studied over many years, the mechanism of activation of ion channels remains a topic of ongoing debate that has not been possible to solve with established experimental methods. Two-dimensional infrared (2D IR) spectroscopy is an emerging analytical technique that probes structural changes of proteins with chemical bond specific spatial and high temporal resolution. Recent technological advances permit applications of 2D IR spectroscopy to ion channels under physiologically realistic conditions. This project combines molecular dynamics simulations with computational spectroscopy to study activation mechanisms of K+ channels. Simulations will be validated by 2D IR experiments and, in turn, will be used to design new experiments that can provide most clear insight into activation mechanisms of K+ channels. The proposed work will derive the atomistic-level description of function of K+ channels. Understanding the mechanisms underlying K+ channels functioning is a key factor in determining the cause of severe diseases such as cardiac arrhythmias and epilepsies. It offers the prospect of designing therapies for ion channel pharmacology. This Research Infrastructure Improvement Track-4 EPSCoR Research Fellows (RII Track-4) project would provide a fellowship to an Assistant Professor at the University of Delaware (UD). Voltage-gated K+ channels (Kv) are integral membrane proteins that selectively conduct K+ ions across cell membranes according to the electrochemical gradient. The voltage-sensing domain (VSD) of Kv senses changes in the membrane electrical potential and triggers a conformational change resulting in the opening and closing of the channel. Despite a wealth of NMR and X-ray crystallography data, many fundamental questions regarding the function of the VSD remain open. It has been hypothesized that the S4 helical segment of the VSD undergoes a conformational and/or hydrational change during voltage-gating, but this hypothesis has never been tested by a direct structural measurement because most biophysical structural techniques cannot be performed under applied voltage. 2D IR spectroscopy can probe protein structures with site-specific resolution and under physiological conditions including applied voltage. It can also probe the gating dynamics of the VSD occurring on the millisecond timescale. Recently the sensitivity of 2D IR spectroscopy has been significantly increased, making it uniquely suitable to study ion channels. The proposed work will combine molecular dynamics simulations with computational 2D IR spectroscopy to elucidate the conformational and hydrational changes of the VSD during voltage activation. The existing structural models of the VSD will be simulated and tested against 2D IR experiments. Based on the comparison with experiments, the models will be refined, or new models will be developed. Simulations will also be utilized to design new 2D IR experiments. We aim to determine the relevant conformations of the VSD and the order they occur during voltage activation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.