Engineering Synthetic Protein Interaction Switches and Memory Modules

• Applicant: Professor Dr. Viktor Stein
• Subject Area: Biochemistry ; Biophysics
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 437166030

A key goal in synthetic biology is to engineer artificial signal transduction circuits to process biomolecular queues in an autonomous and integrated fashion. Applications are manifold ranging from autonomous sensory systems for basic research and biotechnology to reprogramming immune cells with enhanced therapeutic functions. Yet, the construction of such circuits is currently hampered by a lack of suitable components that operate sufficiently independent and in parallel to the endogenous cellular context. In this regard, recent efforts have focussed on turning viral proteases into tailored sensors and signal transducers to confer increasing levels of post-translational control; yet, these systems suffer from slow response times and the type of queues that can be received. Thus, aiming to overcome these limitations, our first goal is to devise a generally applicable approach to construct allosteric and proximity-dependent protein interaction modules that can receive and transmit signals between viral protease transducers and their downstream targets. This will be realised using a combination of structure-guided protein engineering and limited screening focussing on the role of peptide linkers that mediate allosteric transitions and co-operative interactions. Construction efforts will be complemented by detailed biophysical analysis to better understand how peptide linkers mediate switch-like transitions at the molecular level. The second goal is to construct bistable signalling circuits based on a protease toggle switch. Crucially, distinct bistable states will be inducible through post-translational queues and subsequently maintained to provide a form of molecular memory. Bistable circuits are engineered using a combination of structure- and topology-guided design while genetic and environmental parameters for optimal performance are fine-tuned by means of combinatorial high-throughput screening. Finally, circuits will be examined by means of quantitative hysteresis to dissect the molecular factors underlying bistabilty. In addition, a set of degradation specific reporters will shed light which cellular pathways become limiting in the maintenance of bistable states and allow us to evaluate their potential as memory modules to record post-translational queues.

• DFG Programme: Research Grants