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

与折叠蛋白不同，固有无序蛋白(IDPs)在其天然状态下缺乏持久的二级或四级结构。由于他们的动态，国内流离失所者居住在相互转换结构的集合中。这种系综的一个重要特征是它的标度行为，它描述了系综的尺度如何随残基的数量而变化。从聚合物理论的角度来看，标度包含了在给定系综中操作的优先分子相互作用的主旨，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的。从生物学的角度来看，这种伸缩行为在很大程度上使人们能够预测蛋白质的生物学和细胞功能。例如，近年来，对含有大的无序片段的IDPs或蛋白质的液-液相分离的研究呈爆炸性增长。这种相行为通常可以通过聚合物中相分离的形式来解释，而IDP系综的尺寸和比例在这里起着至关重要的作用。用于评估系综构象方面的最常见的实验观测数据是RG(回转半径)，通常由小角X射线散射(SAXS)和RE(端到端距离)测量，由荧光共振能量转移光谱(FRET)测量，特别是单分子smFRET。这两种方法不一致的事实一直是一场持续的辩论，去年在一些有影响力的杂志上发表的关于这一现象的起源的相互矛盾的文章中，这场辩论相形见绌。值得注意的是，这场辩论挑战的是整个领域，而不仅仅是单一的论文。我们提出了一种残基特异性异常SAXS(ASAXS)方法，能够从同一样本中测量RG和RE，并为这一争论提供了最终答案。我们将开发新的遗传密码扩展技术，将微小的异常X射线散射体在完全相同的位置插入到一大组蛋白质中，在smFRET中放置染料标签。这将与先进的工具开发相结合，从基于单原子的散射体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。我们将进一步推进基于ASAXS的新方法，不仅仅是解决IDP的争论，以展示其在动态蛋白质中测量大距离的能力，超出目前任何技术所能达到的规模，从而产生一种新的综合结构生物学工具。

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.