Synthesis and Functional Reconstitution of Light-Harvesting Complex II into Polymeric Membrane Architectures.

One of most important processes in nature is the harvesting and dissipation of solar energy with the help of light-harvesting complex II (LHCII). This protein, along with its associated pigments, is the main solar-energy collector in higher plants. We aimed to generate stable, highly controllable, and sustainable polymer-based membrane systems containing LHCII-pigment complexes ready for light harvesting. LHCII was produced by cell-free protein synthesis based on wheat-germ extract, and the successful integration of LHCII and its pigments into different membrane architectures was monitored. The unidirectionality of LHCII insertion was investigated by protease digestion assays. Fluorescence measurements indicated chlorophyll integration in the presence of LHCII in spherical as well as planar bilayer architectures. Surface plasmon enhanced fluorescence spectroscopy (SPFS) was used to reveal energy transfer from chlorophyll b to chlorophyll a, which indicates native folding of the LHCII proteins.

Nanoscopic leg irons: harvesting of polymer-stabilized membrane proteins with antibody-functionalized silica nanoparticles.

Silica-based nanoparticles (SiNPs) are presented to harvest complex membrane proteins, which have been embedded into unilammelar polymersomes via in vitro membrane assisted protein synthesis (iMAP). Size-optimized SiNPs have been surface-modified with polymer-targeting antibodies, which are employed to harvest the protein-containing polymersomes. The polymersomes mimic the cellular membrane. They are chemically defined and preserve their structural-functional integrity as virtually any membrane protein species can be synthesized into such architecture via the ribosomal context of a cellular lysate. The SiNPs resemble 'heavy leg irons' catching the polymersomes in order to enable gravity-based generic purification and concentration of such proteopolymersomes from the crude mixture of cellular lysates.

Functional Cell Adhesion Receptors (Integrins) in Polymeric Architectures.

Integrins, as transmembrane heterodimeric receptors, have important functions in cell adhesion, migration, proliferation, survival apoptosis and signal transduction, in many physio- as well as pathophysiological settings. Characterisation of integrins and their ligand/antagonist binding is notoriously difficult, due to high integrin redundancy and ubiquity. Bypassing the intrinsic difficulties of cell-based integrin expression, purification and reconstitution, we present for the first time the synthesis of a heterodimeric integrin receptor and its assembly into a block-copolymeric membrane mimic. We present comprehensive data to demonstrate the synthesis of functionally active integrin αv ß3, generated by in vitro membrane-assisted protein synthesis (iMAPS). This work represents the first step towards a robust and adaptable polymer-based platform for characterisation of integrin-ligand interactions.

Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes.

Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.