A genetically encoded ASAXS ruler to study the dimension of intrinsically disordered proteins

基因编码的 ASAXS 标尺，用于研究本质无序蛋白质的尺寸

• 批准号: 432343117
• 负责人: Professor Dr. Edward A. Lemke
• 金额: --
• 依托单位: Außenstelle Hamburg
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/432343117?language=en
• 关键词: genetically; encoded; ASAXS; ruler; study

Intrinsically disordered proteins (IDPs) lack persistent secondary or quaternary structures in their native states unlike folded proteins. Due to their dynamics, IDPs populate ensembles of interconverting structures. An important characteristic of such an ensemble is its scaling behavior describing how do the dimensions of the ensemble scale with the number of residues. From the perspective of polymer theory scaling contains the gist of preferential molecular interactions operative in a given ensemble; that is the scaling exponent suggests whether an ensemble is dominated by self-self-interactions or self-solvent interactions. From a biological standpoint, the scaling behavior largely enables one to predict biological and cellular functions of the proteins. For example, the recent years have seen an explosion of studies of liquid-liquid phase separation of IDPs or proteins bearing large disordered segments. Such phase behavior can often be explained through the formalisms of phase separation in polymers, and the dimensions and scaling of the IDP ensembles play a crucial role here. The most common experimental observables used to assess conformational aspects of the ensembles are RG (radius of gyration) typically obtained from small-angle X-ray scattering (SAXS) and RE (end to end distance) measured from fluorescence resonance energy transfer spectroscopy (FRET), especially single molecule, smFRET. The fact that the two methods disagree has been an ongoing debate, which eclipsed last year in a number of conflicting publications in high impact journals on the origin of this phenomenon. Notably, the debate challenges an entire field, not just single papers. We propose a residue specific anomalous SAXS (ASAXS) approach enabling to measure RG and RE from the same sample and provide the ultimate answer to this debate. We will develop novel genetic code expansion techniques to insert small anomalous X-ray scatters at exactly the same position into a large set of proteins, where in smFRET dye labels are placed. This will be paired with advanced tools development to extract the weak anomalous signal from single atom based scatters, and the ASAXS experiments will be conducted at the arguably world most advanced biological SAXS beamline. We will advance the new ASAXS-based method further beyond just resolving the IDP debate to demonstrate its ability to measure large distances in dynamic proteins beyond scales currently accessible by any technique, thus generating a new integrative structural biology tool.

与折叠蛋白质不同，内部无序蛋白质（IDP）在其天然状态下缺乏持久的二级或四级结构。由于其动态，国内流离失所者居住在相互转换的结构中。这样的集合的一个重要特征是它的标度行为，描述了集合的维度如何随着残基的数量而标度。从聚合物理论的角度来看，标度包含了在给定系综中优先分子相互作用的要点;即标度指数表明系综是由自-自相互作用还是自溶剂相互作用主导。从生物学的角度来看，标度行为在很大程度上使人们能够预测蛋白质的生物和细胞功能。例如，近年来已经看到了对具有大的无序片段的IDP或蛋白质的液-液相分离的研究的爆炸。这种相行为通常可以通过聚合物中相分离的形式来解释，IDP系综的尺寸和缩放在这里起着至关重要的作用。最常见的实验观测用于评估构象方面的合奏是RG（回转半径），通常从小角X射线散射（SAXS）和RE（端到端的距离）测量荧光共振能量转移光谱（FRET），特别是单分子，smFRET。这两种方法不一致的事实一直是一个持续的辩论，去年在高影响力期刊上发表的关于这一现象起源的一些相互冲突的出版物中黯然失色。值得注意的是，这场辩论挑战了整个领域，而不仅仅是单个论文。我们提出了一种残留特定的异常SAXS（ASAXS）的方法，使测量RG和RE从同一个样品，并提供最终的答案，这场辩论。我们将开发新的遗传密码扩展技术，将小的异常X射线散射在完全相同的位置插入到一个大的蛋白质组中，在smFRET染料标签被放置的地方。这将与先进的工具开发相结合，以从基于单原子的散射中提取微弱的异常信号，ASAXS实验将在可以说是世界上最先进的生物SAXS光束线上进行。我们将进一步推进新的基于ASAXS的方法，而不仅仅是解决IDP争论，以证明其能够测量动态蛋白质中的大距离，超出目前任何技术都可以达到的尺度，从而产生一种新的综合结构生物学工具。