Marker Free Protein Analysis System

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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.