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物来执行其特定的任务。这些复合物中的一些随着时间的推移是高度稳定的，而另一些仅仅通过以高速率形成和解离的弱分子键网络保持在一起。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）方法中的“分辨率革命”使这种复合物适合于结构研究，这导致了对其作用机制的前所未有的见解。在这里，我们请求资金用于在维尔茨堡朱利叶斯马克西米利安大学（JMU）生物中心安装一个与多角度光散射检测器（micro-SEC-MALS）耦合的微型尺寸排阻色谱系统，以研究参与各种细胞过程的蛋白质和蛋白质-核酸复合物，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。与以主要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反，（微）SEC-MALS以高精度和速度提供关于大分子复合物的绝对分子量的信息，而不管形状或序列组成如何。它比分析超离心法容易得多，后者需要大量的浓缩样品、成本密集型设备和高度专业的数据分析知识，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;与测量纳摩尔范围内样品的替代方法相反，如质量光度法。所要求的仪器将能够在很宽的尺寸范围内分辨少量的大分子蛋白质和蛋白质-核酸复合物，并以绝对精确度测定其分子量。这些特征是通过将分析（超）高压液相色谱（（U）HPLC）仪器与高灵敏度微体积多角度光散射和折射率（RI）检测器耦合来实现的。温度控制自动进样器的集成允许非常小的样品体积的应用，这通常是有限的大和难以获得的复合物。同时，分析级分收集器的附件允许回收分离的样品用于下游处理，例如cryo-EM，这已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.