Durable vesicles for reconstitution of membrane proteins in biotechnology

Abstract

The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced in vitro performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with.

Keywords: bionanotechnology; block copolymers; in vitro reconstitution; membrane proteins; synthetic biology; vesicles.

Figure 1.. Polymersomes.

Polymer membranes are formed from amphiphilic block copolymers that often have AB, ABA or ABC polymer architectures. These membranes are interdigitated with a viscous polymer melt at its core and a hydrophilic corona of polymers in an extended brush-like conformation. Triblock copolymers may be a mixture of transmembrane and hairpins, which have both their hydrophilic blocks displayed at the same membrane surface. AB and ABA architectures and resulting polymerosome structures are shown on the left-hand side. Asymmetric ABC polymers (right-hand side) can give rise to asymmetric membrane chemistries if the hydrophilic block lengths are different: longer polymers prefer the positive curvature at the exterior of the vesicle and vice versa. Membrane proteins can be inserted into these polymer membranes even if the polymer membrane is much thicker than the hydrophobic thickness of the IMP, suggesting conformational adaption of the polymers to the protein, shown for OmpF (PDB ID: 2OMF) on the left-hand side [30]. Asymmetric ABC membranes may also help drive preferential orientation of the IMPs within the membrane, shown for Aqp0 (PDB ID: 2B6P) on the right-hand side [31].

Figure 2.. Hybrid vesicles.

Hybrid vesicles combine lipids and block copolymers into blended membranes. These hybrid membranes can either be well mixed, giving homogeneous properties across the surface of the vesicle (left-hand side), or phase separated into lipid-rich and polymer-rich domains, which give rise to textured vesicle morphologies with coexisting domains of different structures and properties (right-hand side). Membrane proteins can be inserted into these hybrid membranes, either into homogeneous membranes (left-hand side, showing cyt bo₃; PDB ID: 1FFT) or phase-separated membranes, where the preferred location of the IMP in the membrane is dependent on the relative properties of these two coexisting phases (right-hand side, showing MloK1; PDB ID: 4CHW).