Protein structure and dynamics in ultra-heterogeneous environments

超异质环境中的蛋白质结构和动力学

基本信息

批准号：
10408147
负责人：
Carlos Raul Baiz
金额：
$ 20.58万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-05-31
项目状态：
已结题

项目摘要

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.

概括拥挤和异质性：体内生物分子组织是由拥挤和异质性驱动的。日期、蛋白质结构、动力学和折叠几乎完全在简单的缓冲溶液中进行研究，然而，最近很明显，大多数“试管”研究不能直接转化为细胞研究非特异性静电相互作用、排除体积效应和破坏的氢键。网络决定了这些复杂环境中的蛋白质热力学，而这些的普遍观点。排除体积效应有利于更紧凑的本土国家，我们的小组和其他人一起发现焓贡献增强了蛋白质-水氢键，这些相互作用可以增加主链。因此，迫切需要量化相互作用。目前的研究是迈向精确的类细胞环境中生物分子之间的关键的第一步。了解蛋白质的体内结构和动力学我们的项目旨在表征蛋白质的结构、动力学、以及准确模拟细胞质的拥挤溶液中蛋白质的稳定性。具体来说，我们将量化。分子异质性程度并确定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：钙调蛋白 (CaM) 调节生物通过调节多种蛋白质（包括许多离子通道）的行为来发挥作用。 CaM 调节的离子通道内的突变是导致神经和心血管疾病的原因。可以被认为是多个离子通道的“Ca 感应域”，但 CaM 之间的动态关联离子通道的首次完整结构使得机理研究具有挑战性。 CaM 在今年早些时候（2018 年）得到了解决，这凸显了门控机制仍然存在的事实。例如，膜片钳测量模型需要八种状态，但仅此而已。我们建议通过详细研究门控机制。使用模拟 CaM 结合的肽对动态 CaM 通道相互作用进行生物物理检查 SK2 通道 (KCa2.2) 的结构域在多种生理系统中都很重要。作为理解 Ca2+-CaM 介导的门控的系统，它具有许多优势。如果成功，我们的研究将会实现。产生 CaM 介导的通道激活的逐步机制视图。