Constructing artificial respiratory chain in polymer compartments: Insights into the interplay between bo ₃ oxidase and the membrane

Significance

Analogous to phospholipids, some polymers assemble into vesicles and can mimic cellular membranes. Apart from enabling compartmentalization in the context of artificial cells, amphiphiles may serve as interface for proteins. However, complex transmembrane proteins were reconstituted in polymers with limited success so far. We functionally integrated the proton pump bo ₃ oxidase (part of the bacterial respiratory chain) in synthetic membranes made of PDMS- g-PEO and demonstrated lumen acidification. We provided mechanistic insights into the interplay between the protein and the (semi)synthetic membrane by measuring bending rigidity, lateral diffusion and disorder, proton permeability, and protein partitioning. Polymer and hybrid membranes displayed favorable properties for the construction of artificial cells such as membrane rearrangement, enhanced stability and fluidity, while keeping the compartments proton-tight.

Abstract

Cytochrome bo ₃ ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F ₁F _o-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo ₃ oxidase in giant unilamellar vesicles made of PDMS- g-PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min ⁻¹. To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS- g-PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.