Marker Free Protein Analysis System

对细胞过程的深入机制理解强烈依赖于体外重构方法，包括生化和生物物理方法。将单个反应甚至整个途径从细胞环境中分离出来，可以在分子水平上对其进行表征，而不会受到细胞环境的干扰。为此，在参与本提案的小组中进行的重构方法提供了与蛋白质稳态（伴侣介导的蛋白质折叠，展开和降解）和细胞周期检查点（DNA损伤信号传导，着丝点-微管附着）相关的广泛分子过程的重要见解。这些过程是由多组分系统进行的，其中蛋白质和辅助因子的协调，顺序相互作用提供了管理细胞应激和细胞周期调节。确定在这些多组分系统中定义生物分子相互作用的参数，如亲和常数、接通速率和关闭速率，是提高我们对基本细胞过程理解的关键。生物层干涉法（BLI）是研究生物系统相互作用的最佳方法。BLI能够实时、无标记地分析生物分子相互作用，并精确定量复杂样品中的蛋白质。BLI依赖于测量从生物传感器参考表面到与该表面结合的生物分子的一层光学厚度的变化。具体地说，从两个表面反射的白光的干涉模式的变化被检测到。一个典型的实验是将配体装载到生物传感器的表面上。然后将负载配体的生物传感器浸入含有分析物的溶液中，即不需要微流体，直接将检测表面带到分析物上。分析物与固定化配体结合并引起生物层厚度的变化。这种光学厚度的变化是作为参考表面的波长位移来检测的，并且是绑定的直接测量。给定这个读数，分析物溶液中未结合的分子不会干扰测量。因此，即使是粗裂解物也可用作分析液。利用BLI，我们将能够比较蛋白质网络中的相互作用参数，评估潜在的合作结合模式，并确定生物分子复合物的半衰期。除了表征蛋白质-蛋白质相互作用外，我们计划建立功能分析，例如依赖BLI作为读数的体外泛素化分析。最后，BLI测量将有助于设计和研究小分子调节剂对蛋白质稳态和细胞周期检查点相关反应的影响。

An in-depth mechanistic understanding of cellular processes is strongly dependent on in vitro reconstitution approaches, involving biochemical and biophysical methods. Taking individual reactions or even whole pathways out of the cellular context enables their characterization at the molecular level without confounding effects from the cellular environment. To this end, reconstitution approaches performed in the groups participating in this proposal provided important insight into a wide range of molecular processes related to protein homeostasis (chaperone-mediated protein folding, unfolding, and degradation) and cell cycle checkpoints (DNA damage signalling, kinetochore-microtubule attachment). These processes are carried out by multi-component systems where the coordinated, sequential interaction of proteins and co-factors affords managing cellular stress and cell cycle regulation. Determining parameters that define bio-molecular interactions in these multi-component systems, such as affinity constants, on-rates, and off-rates is key to advance our understanding of essential cellular processes. Bio-layer interferometry (BLI) is optimized for studying interactions in biological systems. BLI enables the real-time, label-free analysis of bio-molecular interactions and accurate quantification of proteins in complex samples. BLI relies on measuring the changes in the optical thickness of a layer spanning from a biosensor reference surface to biomolecules bound to that surface. Specifically, a change in interference pattern of white light reflected from the two surfaces is detected. A typical experiment involves the loading of a ligand onto the surface of a biosensor. The ligand-loaded biosensor is then dipped into a solution containing the analyte i.e. the detection surface is brought directly to the analyte without the need of microfluidics. The analyte binds to the immobilized ligand and causes a change in the thickness of the bio-layer. This change in optical thickness is detected as a wavelength shift from the reference surface and is a direct measure for binding. Given this read-out, un-bound molecules in the analyte solution do not disturb the measurement. Accordingly, even crude lysates can be used as analyte solution. Using BLI we will be able to compare interaction parameters within protein networks, assess potential cooperative binding modes, and determine half-lives of bio-molecular complexes. Beyond characterizing protein-protein interactions, we plan to establish functional assays, such as in vitro ubiquitination assays that rely on BLI as a readout. Finally, BLI measurements will aid in designing and studying the effect of small molecule modulators of protein homeostasis- and cell cycle checkpoint-related reactions.