Exclusion chromatography system coupled to detectors

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 529276114

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.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Größenausschlusschromatographie-System mit Detektoren
• Instrumentation Group: 1350 Flüssigkeits-Chromatographen (außer Aminosäureanalysatoren 317), Ionenaustauscher
• Applicant Institution: Julius-Maximilians-Universität Würzburg
• Leader: Professor Dr. Christian H. Häring