接近溶解度极限的单分散样品的小角X射线散射实验
Small angle x-ray scattering experiments of monodisperse samples close to the solubility limit
摘要：
通过液液相分离（LLPS）将生物分子缩合成生物分子凝聚体是驱动细胞组织的普遍存在的机制。为了实现这些功能，生物分子已经进化为驱动LLPS并促进分配成生物分子凝聚体。确定编码LLPS的蛋白质的分子特征将为过多的生物过程提供重要的见解。有问题的是，直接探测生物分子密相通常在技术上是困难的或不可能的。通过利用生物分子在稀溶液和密相中的构象行为之间的对称性，可以通过对稀相的精确测量来推断对相分离至关重要的细节，从而规避密相的复杂表征。稀相和密相之间的对称性在生物分子的构象系综的大小和形状中被发现-这些参数是小角X射线散射（SAXS）理想地适合于探测的。最近的技术进步使得能够在足够低的浓度下准确地表征内在无序蛋白区域的样品，以避免分子间吸引、寡聚化或聚集的干扰，所有这些都是以前表征自组装蛋白的障碍。在此，我们描述了测量这些样品所固有的陷阱，规避这些问题所需的细节和分析方法，将SAXS测量结果放入LLPS的理论框架中。
英文摘要：
The condensation of biomolecules into biomolecular condensates via liquid-liquid phase separation (LLPS) is a ubiquitous mechanism that drives cellular organization. To enable these functions, biomolecules have evolved to drive LLPS and facilitate partitioning into biomolecular condensates. Determining the molecular features of proteins that encode LLPS will provide critical insights into a plethora of biological processes. Problematically, probing biomolecular dense phases directly is often technologically difficult or impossible. By capitalizing on the symmetry between the conformational behavior of biomolecules in dilute solution and dense phases, it is possible to infer details critical to phase separation by precise measurements of the dilute phase thus circumventing complicated characterization of dense phases. The symmetry between dilute and dense phases is found in the size and shape of the conformational ensemble of a biomolecule - parameters that small-angle x-ray scattering (SAXS) is ideally suited to probe. Recent technological advances have made it possible to accurately characterize samples of intrinsically disordered protein regions at low enough concentration to avoid interference from intermolecular attraction, oligomerization or aggregation, all of which were previously roadblocks to characterizing self-assembling proteins. Herein, we describe the pitfalls inherent to measuring such samples, the details required for circumventing these issues and analysis methods that place the results of SAXS measurements into the theoretical framework of LLPS.
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