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建方法，包括生物化学和生物物理方法。将单个反应甚至整个途径从细胞环境中取出，能够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，在参与该提案的小组中进行的重建方法提供了对与蛋白质稳态（分子伴侣介导的蛋白质折叠，解折叠和降解）和细胞周期检查点（DNA损伤信号传导，激动剂微管附着）相关的广泛分子过程的重要见解。这些过程通过多组分系统进行，其中蛋白质和辅因子的协调、顺序相互作用提供了对细胞应激和细胞周期调节的管理。确定在这些多组分系统中定义生物分子相互作用的参数，例如亲和常数，结合速率和解离速率，是推进我们对基本细胞过程的理解的关键。生物层干涉测量法（BLI）被优化用于研究生物系统中的相互作用。BLI能够实时、无标记地分析生物分子相互作用，并准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结合到该表面的生物分子的层的光学厚度的变化。具体地，检测从两个表面反射的白色光的干涉图案的变化。典型的实验涉及将配体加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，即检测表面直接接触分析物而不需要微流体。分析物与固定化配体结合并引起生物层厚度的变化。光学厚度的这种变化被检测为相对于参考表面的波长偏移，并且是结合的直接量度。给定该读出，分析物溶液中未结合的分子不会干扰测量。因此，甚至粗裂解物也可以用作分析物溶液。使用BLI，我们将能够比较蛋白质网络内的相互作用参数，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-蛋白质相互作用之外，我们计划建立功能测定，例如依赖BLI作为读数的体外遍在蛋白化测定。最后，BLI测量将有助于设计和研究蛋白质稳态和细胞周期检查点相关反应的小分子调节剂的作用。

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.