Designing interactions mediated by disordered protein regions

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Proteins are the principle constituents of cells, and they are involved in nearly all processes that take place in cells. Key to their contributions are interactions that regulate cellular functions and transmit signals. In the last three decades, tremendous progress has been made in the structural and functional characterization of intra and intermolecular protein interactions. Moreover, computational and experimental protein engineering tools have been developed that enable us to design and select for new proteins with desired interaction features. This progress has been particularly impactful for "classical" proteins that fold into well-defined tertiary structures. However, many proteins contain large intrinsically disordered regions (IDRs). For these generally more dynamic protein elements, progress in structural characterization and functional reengineering has been much more modest. Given the important role IDRs play in allosteric and steric activity regulation as well as dynamic assembly of pivot proteins in regulatory and signaling networks, it is clear that we need to develop new tools that allow us better understand IDR-mediated interactions as well as engineer IDRs with desired interaction properties. In this research program, we propose to employ a combination of computational and experimental approaches in order to set up new pipelines for the successful engineering of IDRs with desired interaction properties. In our first aim, we will develop deep learning-based tools for the identification of IDRs that, via transient intramolecular interactions, act as switches of protein activity. Taking advantage of these new tools and large-scale, molecular dynamics (MD) simulation-based mutation screens, we will then design transcription factors that have IDRs with specific switch behaviour. In the second aim, we will use a flow cytometry-based screening method in combination with deep learning to identify which sequence features confer phase-separation capabilities to IDRs, respectively, the ability to locate to stress granules. We will exploit what has been learned to design IDRs with tunable phase separation behaviors, i.e. desired intermolecular interaction properties. The research program proposed here aims to open up new avenues for the exploitation of the recently evolved and very versatile IDRs, which will have significant impact on our ability to design interactions within and between proteins and thus steer specific cellular processes for applications in academia and industry.