Beyond mere flexibility: Functional Peptide Linkers in Engineered Nanosensors and -switches

• Main Author: Gräwe, Alexander
• Format: Others
• Language: en
• Published: 2020
• Online Access: https://tuprints.ulb.tu-darmstadt.de/14535/19/Dissertation_Graewe_092020.pdf; Gräwe, Alexander <http://tuprints.ulb.tu-darmstadt.de/view/person/Gr=E4we=3AAlexander=3A=3A.html> (2020): Beyond mere flexibility: Functional Peptide Linkers in Engineered Nanosensors and -switches. (Publisher's Version)Darmstadt, Technische Universität, DOI: 10.25534/tuprints-00014535 <https://doi.org/10.25534/tuprints-00014535>, [Ph.D. Thesis]

Human health monitoring, disease diagnosis and therapeutics rely on the detection of biomolecules. In this regard, Synthetic Biology approaches based on tailored molecular receptors and actuators can create powerful biosensing platforms. To achieve an optimal biosensor, the composition of such receptor/actuator scaffolds requires both careful design and thorough screening. Thus, my PhD studies focused on construction principles that render modular fusion proteins powerful biosensors. Central to the functionality of fusion proteins are the domain-connecting peptidic linkers. While the importance of linkers is known, methods to systematically screen the underlying amino acid space are scarce. Therefore, a novel DNA assembly method was devised that enables straightforward cloning of large and diverse linker libraries. By applying the strategy to synthetic protein switches, I identified multiple potent ligand-responsive proteases. The importance of linkers was further assessed by investigating the behavior of nanopore scaffolds based on the β-barrel transmembrane protein FhuA ΔcΔ5L. The linker between the transmembrane domain and engineered terminal receptor tags emerged as a crucial parameter, impacting both open probability and intermolecular interaction ability of the nanopore in artificial lipid bilayers. An engineered ΔcΔ5L variant could irreversibly catch a second fusion protein while embedded into the bilayer, demonstrating biosensing at the single molecule level. While biological nanopores are highly specific, their lack of stability complicates their use in application-oriented biosensors. Considering this, a strategy to stably immobilize fusion protein receptors in solid-state nanopores was developed in collaboration with Ivana Duznovic from the Materials analysis group. Highly specific nanobodies attached to conical nanopores in track-etched poly(ethylene terephthalate) (PET) membranes allowed sensitive discrimination of analytes by current-voltage (I-V) measurements. The developed nanobody-nanopore platform constitutes a highly modular biosensing system and can potentially be combined with lab-on-chip devices. In the first chapter, I embed my dissertation in the context of Synthetic Biology and Nanosensors while outlining the content of the following chapters. The second chapter describes the development of a cloning strategy for protein linkers and its application to synthetic protein switches. The third chapter deals with the biological nanopore scaffold ΔcΔ5L, while the fourth chapter describes the development of solid-state nanopore biosensing platform.