Protein stabilizers from tardigrades

项目负责人/主要研究者（最后，第一，中间）：Pielak，加里J。 项目摘要。 水熊虫是一种微小的动物，能够在极端的压力下生存，包括干燥，冷冻，煮沸， 大剂量的电离辐射，甚至外层空间的真空。我们已经确定了蛋白质家族 使水熊虫能够在干燥的环境中存活。这些胞质丰富的热溶性（CAHS）蛋白是 是缓步动物特有的该项目的目标是确定CAHS蛋白质使用的保护机制， 保护客户蛋白免受干燥诱导的损伤。这些知识将有助于开发新的 分子，以增加基于蛋白质的药物和工业酶的稳定性和保质期。 我们假设CAHS蛋白通过在可逆基质中的包封来保护客户蛋白。我们 提出了一个模型，其中完全水合的CAHS蛋白具有部分折叠的末端， 柔性连接器除去水后，末端分子间相互作用，在周围形成保护性凝胶基质。 客户端蛋白质即使在完全干燥后，基质也能保持并保持客户蛋白质保护。 该模型得到了初步发表的和新的数据，从酶活性的研究，核磁共振检测 酰胺质子交换，19 F核磁共振，圆二色谱，傅里叶变换红外 光谱学和流变学。该模型将在三个独立和互补的目标中进一步测试。 目标1：确定固态中的序列/功能关系。我们预测保护是模块化的;连接器 可以交换，并且只要每个分子具有被一个 连接器。我们将使用由单独的模块和模块组合组成的片段来测试这个想法。我们 将决定CAHS蛋白及其片段保护酶免受干燥的程度- 诱导的失活，并评估它们通过以下方式保持固态模型蛋白质结构的能力： 使用NMR检测的酰胺质子/氘交换。 目的2：确定凝胶和溶液中的序列/功能关系。我们将量化CAHS的影响 蛋白质和片段对客户蛋白质的解折叠的修饰的标准状态自由能的影响。 目标3：定义固态、凝胶和溶液中的序列/结构关系。我们将量化 的CAHS蛋白质和它们的片段形成凝胶使用流变仪，一种方法来量化凝胶强度。的 完整CAHS蛋白及其片段的二级结构将使用同步辐射进行定量 圆二色谱偏振光谱法和傅里叶变换红外光谱法。 OMB编号0925-0001/0002（2016年3月修订版，批准至2018年10月31日）

Program Director/Principal Investigator (Last, First, Middle): Pielak, Gary J. Project Summary. Tardigrades are microscopic animals that survive extreme stresses, including desiccation, freezing, boiling, large doses of ionizing radiation and even the vacuum of outer space. We have identified the family of proteins that allows tardigrades to survive desiccation. These Cytosolic Abundant Heat Soluble (CAHS) proteins are unique to tardigrades. The goal of the project is to define the protective mechanism used by CAHS proteins to shield client proteins from desiccation-induced damage. This knowledge will facilitate the development of new molecules to increase the stability and shelf life of protein-based drugs and industrial enzymes. We hypothesize that CAHS proteins protect client proteins via encapsulation in a reversible matrix. We propose a model where fully hydrated CAHS proteins possess partially-collapsed termini separated by a flexible linker. Upon water removal, the termini interact intermolecularly, forming a protective gel matrix around the client proteins. The matrix persists and maintains client protein protection even upon complete desiccation. The model is supported by preliminary published and new data from studies of enzyme activity, NMR-detected amide proton exchange, 19F NMR, circular dichroism spectropolarimetry, Fourier-transform infrared spectroscopy and rheometry. The model will be further tested in three independent and complementary aims. Aim 1: Determine sequence/function relationships in the solid state. We predict that protection is modular; linkers can be swapped, and ends can be swapped as long as each molecule possesses two ends separated by a linker. We will test this idea using fragments comprising the modules alone and combinations of modules. We will determine the degree to which CAHS proteins and their fragments protect enzymes against desiccation- induced inactivation and assess their ability to preserve the structure of a model protein in the solid state by using NMR-detected amide proton/deuterium exchange. Aim 2: Determine sequence/function relationships in gels and solution. We will quantify the effects of CAHS proteins and the fragments on their modified standard-state free energy of unfolding of a client protein. Aim 3: Define sequence/structure relationships in the solid state, gels and solution. We will quantify the ability of CAHS proteins and their fragments to form gels using rheometry, a method to quantify gel strength. The secondary structure of intact CAHS proteins and their fragments will be quantified using synchrotron-radiation circular-dichroism spectropolarimetry and Fourier-transform infrared spectroscopy. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page