Protein structure and dynamics in ultra-heterogeneous environments

摘要 拥挤和异质性：体内的生物分子组织是由拥挤和异质性驱动的。至 数据、蛋白质结构、动力学和折叠几乎都是在简单的缓冲溶液中研究的， 然而，最近有证据表明，大多数“试管”研究不能直接转化为细胞。 环境。非特异性静电相互作用、排除的体积效应和破坏的氢键 在这些复杂的环境中，网络决定了蛋白质的热力学。虽然来自这些国家的主流观点 排除体积效应有利于更紧密的原生状态，我们的团队和其他人发现 焓贡献增强了蛋白质-水的氢键。这些相互作用可以增加主干 暴露，从而破坏折叠状态的稳定。因此，迫切需要量化相互作用 在精确的细胞状环境中的生物分子之间。目前的研究是实现以下目标的关键第一步 了解体内蛋白质的结构和动力学。我们的项目的目的是描述结构，动力学， 和蛋白质在拥挤的溶液中的稳定性，准确地模拟细胞质。具体地说，我们将量化 分子的异质性程度以及大分子拥挤对蛋白质和蛋白质的作用 蛋白质-水接触。 蛋白质-蛋白质相互作用和离子通道门控机制：钙调素(CaM)调节生物 通过调节包括许多离子通道在内的多种蛋白质的行为来发挥作用。CAM突变或 CaM调节的离子通道内的突变与神经和心血管疾病有关。凸轮 可以认为是一个钙敏感结构域，对于多个离子通道，但CaM之间的动态关联 而离子通道使机械学研究具有挑战性。离子通道的第一个完整结构 今年早些时候(2018年)解决了CAM问题。这些都强调了这样一个事实，即门控机制仍然 不完全理解。例如，需要八种状态来模拟膜片钳测量，但仅 已知有两种结构(打开/关闭)。我们建议通过一个详细的 利用模拟CaM结合的多肽对动态CaM-通道相互作用的生物物理检测 SK2通道的域(KCa2.2)。SK通道在多种生理系统中都很重要 作为一种理解钙离子-CaM介导的门控的系统，提供了许多优点。如果成功，我们的研究将 对CaM介导的通道激活产生一种循序渐进的机械观点。

SUMMARY Crowding and heterogeneity: Biomolecular organization in vivo is driven by crowding and heterogeneity. To date, protein structure, dynamics, and folding have been studied almost exclusively in simple buffer solutions, yet it is has recently become evident that most “test tube” studies cannot be directly translated to cellular environments. Nonspecific electrostatic interactions, excluded volume effects, and disrupted hydrogen-bond networks dictate protein thermodynamics in these complex environments. While the prevailing view from these is that excluded-volume effects favor the more compact native states, our group, along with others, found that enthalpic contributions strengthen protein-water hydrogen bonds. These interactions can increase backbone exposure and consequently destabilize folded states. Thus, there is an immediate need to quantify interactions between biomolecules in accurate cell-like environments. The present studies are critical first step towards understanding protein structure and dynamics in vivo. Our project aims to characterize the structure, dynamics, and stability of proteins in crowded solutions that accurately mimic the cytoplasm. Specifically, we will quantify the degree of molecular heterogeneity and establish the role of macromolecular crowding on protein-protein and protein-water contacts. Protein-protein interactions and ion channel gating mechanisms: Calmodulin (CaM) regulates biological function by modulating the behavior of a wide range of proteins including many ion channels. CaM mutations or mutations within CaM-regulated ion channels are responsible for neurological and cardiovascular diseases. CaM can be considered a “Ca-sensing domain” for multiple ion channels, but the dynamic association between CaM and ion channels make mechanistic studies challenging. The first complete structures of an ion channel with CaM were solved earlier this year (2018). These underscore the fact that the gating mechanisms remain incompletely understood. For example, eight states are required to model patch clamp measurements, but only two structures (open/closed) are known. We propose to investigate gating mechanisms through a detailed biophysical examination of dynamic CaM-channel interactions using a peptide that mimics the CaM binding domain of the SK2 channel (KCa2.2). SK channels are important in a wide variety of physiological systems and offer many advantages as a system for understanding Ca2+-CaM-mediated gating. If successful, our studies will produce a stepwise mechanistic view of CaM-mediated channel activation.