Massive parallel de novo design of biological nanopores.

Proteins embedded in membranes play key roles in maintaining cell integrity, homeostasis and communication. Emerging technologies (nanopore sequencing, synthetic cells, …) imitate biological systems and repurpose membrane protein for the transport and sensing of new analytes through synthetic membranes. These applications have fueled the demand for (synthetic) membrane proteins with properties and functions not observed in nature. Structure-based computational protein design is revolutionizing many aspects of biotechnology but has almost exclusively focused on protein folding in water. Our aim is to develop innovative experimental and computational methods to enable the design of transmembrane β-barrels (TMBs), a class of membrane proteins with excellent properties to act as nanopore sensors. My lab was first to demonstrate the feasibility of TMB design and has established a pipeline - from computation to electrophysiology and biochemical characterization. Yet, experimental characterization of designer membrane proteins remains challenging. The exact reasons of success and failure are often occluded by the lack of in cellulo expression of proteins that fail to engage into the biological folding pathways. Additionally, the experimental throughput remains much lower than what can be achieved for water-soluble proteins. Here, we will setup the basis for (i) cell-free experimental characterization of designer TMBs and (ii) a platform for massive parallel design of membrane proteins. This project has all the components to translate into transformative advances in nanopore sensing and sequencing by providing the nanopore R&D community with accurate and innovational computational design methodologies.