Reconstitution of Respiratory Enzymes in PDMS-g-PEO Polymer and Polymer/Lipid Hybrid Vesicles

Abstract

Mechanical characteristics (e.g. rigidity, tightness) of cellular compartments are alterable by partial or complete replacement of natural building blocks with synthetic alternatives, but often at the expense of other properties (e.g. fluidity, thickness). Since the scope of bottom-up synthetic biology aims to reconstitute essential life processes such as selective transport and energy transduction, we seek to retain properties of the interfaces, necessary for interaction with membrane proteins. PDMS-g-PEO polymer appears to accommodate these demands. Thus, we co-reconstituted the chemically-driven proton pump bo3 oxidase and F1Fo-ATPase in PDMS-g-PEO polymer and hybrid nanocompartments (LUVs) in order to build a synthetic energy conversion module. Successful scale-up to the micron-scale plays a crucial role in the real-time visualization and characterization of this module. Via an optimized fusion/electroformation approach we integrated bo3 oxidase in giant vesicles (GUVs) and determined its influence on mechanical properties. The reconstituted enzymes had preserved activity and exhibited unidirectional orientation, which led to the acidification of the synthetic compartments. The most remarkable finding is that the characteristics of hybrid membranes are not always intermediate between pure lipid- and pure polymer ones. Blending led to increased permeability, but after inserting the proton pump the compartments were surprisingly re-sealed. The measurements of active proton pumping and passive proton permeability were done in a microfluidic setup, which enabled monitoring of individual GUVs as well as better control of the experimental conditions. Moreover, we observed an interesting phenomenon beneficial for crowded membranes: While insertion of bo3 oxidase in soy PC decreased the fluidity, it exercised the opposite effect on the polymer by loosening its structure. The F1Fo-GUVs were prepared by the aforementioned procedure and the enzymatic activity was analyzed via outward proton pumping. Finally, respiratory-driven energy-converting GUVs were constructed. Sponsored by BMBF and MPG.