A new perspesective on ion conductance and structural dynamics of ion channels using 2D IR

摘要 自20世纪70年代以来进行的膜片钳实验提供了开放和关闭的时间尺度 离子通道。在过去的20年里，X射线晶体学已经产生了高分辨率的离子结构， 渠道到目前为止，还没有关于通道动力学或施加电压对离子的影响的直接实验。 渠道结构。在这个建议中，我们通过利用新的技术进步将动态与结构联系起来 其使得能够对离子通道进行二维红外（2D IR）测量。2D固有的时间分辨率 红外光谱是几皮秒-比离子通道的毫秒运动快得多。结构 拆分是由于主链羰基振动和静电荷之间的耦合， 离子。残基特异性到结构域特异性的结构解析通过常规同位素标记获得， Valiyaveetil实验室开发的半合成程序。而且，二维红外光谱现在可以计算非常 准确地从短分子动力学轨迹，使实验之间的一对一的比较 和结构或提出的结构模型。二维红外、半合成和分子动力学的结合 模拟允许对离子通道结构动力学的新视角。在这份提案中，我们讨论了两个问题， 在钾离子通道社区的突出争议。在目标1和2中，我们研究了 有争议的新模型的离子渗透通过选择性过滤器的KcsA和NaK，称为“硬敲” 模型硬撞击模型似乎可以解释X射线数据和所有其他现有的测量结果，甚至 尽管它与教科书中最初的“连锁反应”模型根本不一致。在目标3中，我们电压- 触发KvAP通道的电压感应域（VSD）的结构运动，以研究 假设VSD中的基本TM 4螺旋在治疗期间经历构象和/或水化变化， 电压门控，如前所述，但从未通过直接结构或时间分辨 测量.这些目标为离子通道中的突出问题提供了新的科学见解 建立了研究离子通道结构动力学的新技术。

Abstract Patch clamp experiments performed since the 1970s have provided the timescales for the opening and closing of ion channels. X-ray crystallography over the past 2 decades has yielded high-resolution structures of ion channels. As yet, there are no direct experiments on channel dynamics or the effect of an applied voltage on ion channel structures. In this proposal, we connect dynamics to structure by leveraging new technological advances that enable two-dimensional infrared (2D IR) measurements on ion channels. The inherent time-resolution of 2D IR spectroscopy is a few picoseconds – much faster than the millisecond motions of ion channels. Structural resolution arises from couplings between the backbone carbonyl vibrations and electrostatic charges such as ions. Residue-specific to domain-specific structural resolution is obtained with isotope labeling made routine by semisynthesis procedures developed in the Valiyaveetil lab. And, 2D IR spectra can now be calculated very accurately from short molecular dynamics trajectories, enabling a one-to-one comparison between experiment and structure or proposed structural models. This combination of 2D IR, semisynthesis, and molecular dynamics simulations permit a new perspective on ion channel structural dynamics. In this proposal, we address two outstanding controversies in the potassium ion channel community. In Aims 1 and 2, we investigate a controversial new model for ion permeation through the selectivity filter of KcsA and NaK, called the “hard-knock” model. The hard-knock model appears to explain the X-ray data and all other existing measurements, even though it is fundamentally at odds with the original “knock-on” model found in textbooks. In Aim 3, we voltage- trigger the structural motions of the voltage sensing domain (VSD) of the KvAP channel, to investigate the hypothesis that the essential TM4 helix in the VSD undergoes a conformational and/or hydrational change during voltage gating, as previously proposed but never established by a direct structural or time-resolved measurement. These aims provide new scientific insights into outstanding problems in the ion channel community and establish a new technique for studying ion channel structural dynamics.