Exclusion chromatography system coupled to detectors

在过去的十年中，越来越清楚的是，大多数细胞蛋白不是孤立地起作用，而是与其他蛋白质和/或核酸组装成大分子复合物来执行它们的特定任务。随着时间的推移，这些复合物中的一些非常稳定，而另一些仅仅是通过一个弱分子键网络结合在一起，这些网络以高速率形成和解离。深入了解动态蛋白质复合物的组装仍然具有挑战性，需要提供准确和定量读数的方法。与此同时，低温电子显微镜（cryo-EM）方法的“分辨率革命”使得这种复合物适合于结构研究，这使得人们对其作用机制有了前所未有的了解。在这里，我们申请资金用于安装微型尺寸的排除色谱系统耦合多角度光散射检测器（micro-SEC-MALS）在朱利叶斯·马克西米利安大学(JMU) <s:1> rzburg生物中心，以研究蛋白质和蛋白质-核酸复合物参与各种细胞过程，包括基因组组织，基因表达，剪接，蛋白质质量控制和蛋白质降解。与以定性方式测定蛋白质-蛋白质和蛋白质-核酸相互作用的经典方法相比，（micro-）SEC-MALS以高精度和快速的方式提供有关大分子复合物绝对分子质量的信息，而不考虑其形状或序列组成。它比分析性超离心要容易得多，后者需要大量浓缩样品、成本密集型设备和高度专业化的数据分析知识，并且可以在检测弱（微摩尔亲和）相互作用所需的浓度范围内进行；相反的替代方法，测量样品在纳摩尔范围内，如质量光度法。所要求的仪器将能够在大尺寸范围内解析少量大分子蛋白质和蛋白质-核酸复合物，并以绝对精度确定其分子质量。这些特性是通过将分析（超）高压液相色谱（(U)HPLC）仪器与高灵敏度的微体积多角度光散射和折射率（RI）探测器耦合来实现的。温控自动进样器的集成允许应用非常小的样本量，这通常限制了大型和难以获得的复合物。同时，附着的分析组分收集器允许回收分离的样品进行下游处理，例如低温电镜，这已经成为我们研究小组确定大分子复合物结构的首选方法。

Over the past decade, it has become increasingly clear that most cellular proteins do not function in isolation, but instead assemble into large macromolecular complexes with other proteins and/or nucleic acids to perform their specific tasks. Some of these complexes are highly stable over time, while others are held together merely by a network of weak molecular bonds that form and dissociate at high rates. Gaining insights into the assembly of dynamic protein complexes has remained challenging and requires approaches that provide accurate and quantitative readouts. In parallel, the ‘resolution revolution’ in cryo-electron microscopy (cryo-EM) methods has made such complexes amenable for structural studies, which has resulted in unprecedented insights into their mechanisms of action. Here, we request funds for the installation of a microscale size exclusion chromatography system coupled to multi-angle light scatting detectors (micro-SEC-MALS) at the Biocenter of the Julius Maximilian University (JMU) Würzburg to study protein and protein-nucleic acid complexes involved in various cellular processes, including genome organization, gene expression, splicing, protein quality control, and protein degradation. In contrast to classical approaches that assay protein-protein and protein-nucleic acid interactions in a largely qualitative manner, (micro-)SEC-MALS delivers information about the absolute molecular mass of macromolecular complexes with high accuracy and speed, irrespective of shape or sequence composition. It is considerably easier to perform than analytical ultracentrifugation, which requires large volumes of concentrated sample, cost-intensive equipment, and highly specialized knowledge in data analysis, and it can be performed in a concentration range required for the detection of weak (micromolar affinity) interactions; contrary to alternative methods that measure samples in the nanomolar range, such as mass photometry. The requested instrument will be able to resolve small quantities of macromolecular protein and protein-nucleic acid complexes over a wide size range and determine their molecular masses with absolute precision. These features are achieved by coupling an analytical (ultra-)high-pressure liquid chromatography ((U)HPLC) instrument to highly sensitive micro-volume multi-angle light scattering and refractive index (RI) detectors. The integration of a temperature-controlled autosampler allows the application of very small sample volumes, which are often limited for large and difficult-to-obtain complexes. At the same time, the attachment of an analytical fraction collector allows recovery of separated samples for downstream processing, such as cryo-EM, which has become the method of choice for determining structures of macromolecular complexes in our research groups.