Protein structure and dynamics in ultra-heterogeneous environments

总结 拥挤和异质性：生物分子在体内的组织是由拥挤和异质性驱动的。到 日期，蛋白质结构，动力学和折叠几乎只在简单的缓冲溶液中进行研究， 然而，最近变得明显的是，大多数“试管”研究不能直接转化为细胞研究， 环境.非特异性静电相互作用、排除体积效应和破坏氢键 网络决定了蛋白质在这些复杂环境中的热力学。虽然这些人的普遍看法是， 排除体积效应有利于更紧凑的原生状态，我们的团队沿着其他人发现， 水的贡献加强蛋白质-水氢键。这些相互作用可以增加骨架 暴露并因此使折叠状态不稳定。因此，迫切需要量化相互作用 在精确的细胞样环境中的生物分子之间。目前的研究是关键的第一步， 了解体内蛋白质结构和动力学。我们的项目旨在描述结构，动力学， 以及蛋白质在拥挤溶液中的稳定性，这些溶液精确地模拟了细胞质。具体来说，我们将量化 分子异质性的程度，并建立大分子拥挤对蛋白质-蛋白质和 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）调节生物学 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是神经和心血管疾病的原因。凸轮 可以被认为是多个离子通道的“钙敏感域”，但钙调素之间的动态关联 和离子通道使得机理研究具有挑战性。第一个完整的离子通道结构， CaM在今年早些时候（2018年）得到解决。这些都强调了门控机制仍然存在的事实， 不完全理解。例如，需要八个状态来对膜片钳测量进行建模，但是仅需要八个状态。 已知有两种结构（开放/封闭）。我们建议通过一个详细的 使用模拟CaM结合的肽进行动态CaM通道相互作用的生物物理检查 SK2通道（KCa2.2）。SK通道在多种生理系统中是重要的， 作为理解Ca 2 +-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 产生钙调素介导的通道激活的逐步机制的观点。

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.