Pores that Perform: Functionalizing Synthetic Membranes via Engineered Self-Inserting Nanopores

Bobkova, Elisabeth

Thesis

Open Access

Pores that Perform: Functionalizing Synthetic Membranes via Engineered Self-Inserting Nanopores

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Date

2025-10-29

Authors

Bobkova, Elisabeth

Abstract

Biomembranes present an essential encapsulating barrier for living cells. Equipped with a variety of biochemical molecules like lipids, sugars, and membrane proteins, they contribute to a vast array of important cellular functions, including signal transduction, selective nutrient uptake, waste removal, and cell anchoring. In recent years, synthetic membrane systems mimicking some of these biological functions have gained traction in commercial biotechnological applications and synthetic cell engineering. The most successful applications include nanopore sequencing and sensing, which are currently paving the way for commercial biomembrane-based solutions. Given the unique role that membranes hold in biology, further engineering of their synthetic counterparts promises to unlock even more exciting applications down the road, including controlled biochemical reaction separation, targeted therapeutic delivery, or bio-inspired filtration systems. However, the synthetic cell engineering and membrane functionalization fields both currently lack fast, versatile, and reliable protein insertion and screening methods. One promising but underexplored tool to bridge this gap could lie in the use of self-inserting nanopores. These proteins, whose structure has evolved to self-insert into biological membranes to form structurally consistent nanometer-wide pores with a defined internal biophysical environment, are already being exploited for transport and sensing capabilities. Combining these structures with site-specific modifications could expand their functionality in a plug-and-play manner and create programmable and responsive membrane systems. In this work, I thus aim to explore the use of self-inserting nanopores to create a scalable, adaptable, and generalizable platform for introducing novel functionality on synthetic membranes. Specifically, I want to focus on engineering the selective diffusion of small and biologically relevant molecules across the membrane to enable biological reaction separation. As a secondary goal, I also aim to explore the use of functionalized nanopores for membrane display applications. Another key objective of this thesis is to address the notable lack of high- and medium-throughput approaches for probing membrane-associated functions in an in vitro context. I start by creating fusion constructs with peptide-linkers at the N-terminus, alongside pore variants carrying cysteine residues at pore entry and pore lumen, to enable plug and play site-specific modifications. For the latter, I leverage three functionalization strategies: First, I probe the methanethiosulfionate reagents, which selectively react with cysteines under mild conditions and form a reversible modification. The variety of reagents allows for the introduction of positive and negative charges, as well as bulky aromatic residues and backbones for further reactions. Second, I test the cysteine peptide modification to increase the interaction surface with the passing cargo and produce reversible and irreversible modification options. Third, I use maleimide-conjugated peptides, which can selectively react with the cysteine residue, forming a stable, nonreversible modification. With these methods, I show successful modification of nanopores pre- and post-insertion into synthetic membranes at two structurally different sites of 𝛼-Hemolysin and voltagedependent anion-selective channel 1. I compare the two engineered pore types as well as their two modification sites, look at the influence of the modifications on the ion flow at the single-pore level, and explore the changes in the nanopore-mediated diffusion of molecules across the membrane. I further test the use of fusion constructs for membrane display purposes. In addition, I assess and benchmark existing and develop new medium-throughput transport assays to enable reliable screening of synthetic membranes with defined and tunable properties. Specifically, I use the developed nanopore constructs to showcase and compare large unilamellar vesicle-based diffusion assays for divalent ions, small molecule dyes, and the newly developed peptide diffusion assay. I test the compatibility of the assays with different chemicals involved in protein functionalization and discuss the strengths and weaknesses of the different approaches, particularly stressing the importance of integrated breakage controls for probing membrane functions at scale. Overall, through theoretical pore-design considerations, modular nanopore engineering, and biomolecule transport assay prototyping, this thesis aims to expand the field of synthetic biology with a versatile toolbox to engineer and probe membrane functionality.