Exclusion chromatography system coupled to detectors

在过去的十年里，越来越清楚的是，大多数细胞蛋白质并不是孤立地发挥作用，而是与其他蛋白质和/或核酸组装成大分子复合体，以执行其特定的任务。这些络合物中的一些随着时间的推移高度稳定，而另一些仅仅是通过以高速率形成和解离的弱分子键网络保持在一起的。获得对动态蛋白质复合体组装的洞察仍然具有挑战性，需要提供准确和定量读数的方法。同时，低温电子显微镜(Cryo-EM)方法的“分辨率革命”使这类络合物适合进行结构研究，这使人们对它们的作用机制有了前所未有的了解。在此，我们申请资金用于在瑞士马克西米利安大学(JMU)维尔茨堡生物中心安装与多角度光散射检测器(MicroSEC-MALS)耦合的微型尺寸排除层析系统，以研究参与各种细胞过程的蛋白质和蛋白质-核酸复合体，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。与主要以定性方式分析蛋白质-蛋白质和蛋白质-核酸相互作用的经典方法不同，(微)SEC-MALS以高精度和高速度提供关于大分子复合体的绝对分子质量的信息，而不考虑形状或序列组成。它比分析超速离心法容易得多，后者需要大量的浓缩样品、成本密集的设备和高度专业化的数据分析知识，并且可以在检测弱(微摩尔亲和力)相互作用所需的浓度范围内进行；与在纳摩尔范围内测量样品的替代方法(如质量光度法)相反。所要求的仪器将能够在很大的尺寸范围内分解少量的大分子蛋白质和蛋白质-核酸复合体，并绝对精确地测定它们的分子质量。这些功能是通过将分析(超)高压液相色谱(U)仪器与高灵敏度的微体积多角度光散射和折射率(RI)检测器耦合实现的。集成温度控制的自动取样器允许应用非常小的样品体积，而对于大而难以获得的络合物，这往往是有限的。同时，分析级分收集器的连接允许回收分离的样品进行后续处理，如冷冻-EM，这已成为我们研究小组确定大分子络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.