Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents

Abstract

Giant unilamellar vesicles (GUVs) are convenient biomimetic systems of the same size as cells that are increasingly used to quantitatively address biophysical and biochemical processes related to cell functions. However, current approaches to incorporate transmembrane proteins in the membrane of GUVs are limited by the amphiphilic nature or proteins. Here, we report a method to incorporate transmembrane proteins in GUVs, based on concepts developed for detergent-mediated reconstitution in large unilamellar vesicles. Reconstitution is performed either by direct incorporation from proteins purified in detergent micelles or by fusion of purified native vesicles or proteoliposomes in preformed GUVs. Lipid compositions of the membrane and the ionic, protein, or DNA compositions in the internal and external volumes of GUVs can be controlled. Using confocal microscopy and functional assays, we show that proteins are unidirectionally incorporated in the GUVs and keep their functionality. We have successfully tested our method with three types of transmembrane proteins. GUVs containing bacteriorhodopsin, a photoactivable proton pump, can generate large transmembrane pH and potential gradients that are light-switchable and stable for hours. GUVs with FhuA, a bacterial porin, were used to follow the DNA injection by T5 phage upon binding to its transmembrane receptor. GUVs incorporating BmrC/ BmrD, a bacterial heterodimeric ATP-binding cassette efflux transporter, were used to demonstrate the protein-dependent translocation of drugs and their interactions with encapsulated DNA. Our method should thus apply to a wide variety of membrane or peripheral proteins for producing more complex biomimetic GUVs.ABC transporter | DNA transfer | membrane protein

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

“…35,36 In the case of water transport over the bilayer, 37 permeability can also be assessed by monitoring a change in size of the droplets on either side of the interdroplet bilayer. To illustrate the latter application, we created an array of three droplets, with a centre droplet of 4 μL volume containing 0.25 M KCl and two flanking 2 μL droplets with 1 M KCl.…”

Building Model Membranes with Lipids and Proteins: Dangers and Challenges

“…In a recent development of this method, Dezi and coworkers (Dezi et al 2013) have adapted this method to allow the asymmetric incorporation of proteins into giant vesicles. This is important, since as mentioned above, heterogeneity is a point of concern and the use of visible objects allows the heterogeneity to be controlled and vesicles to be assessed individually.…”

“…This is important, since as mentioned above, heterogeneity is a point of concern and the use of visible objects allows the heterogeneity to be controlled and vesicles to be assessed individually. Furthermore, many experiments require being able to visualize the vesicles; this is the case with many of the example measurements made by Dezi et al (2013).…”

Visualization and Quantification of Transmembrane Ion Transport into Giant Unilamellar Vesicles

“…Fluorescence microscopy of GUVs has been used to study passive diffusion of peptides [10] and organic compounds [11] through membranes, passive diffusion of dyes through pores formed by peptides [12] or proteins [13] and to qualitatively study the presence and performance of membrane proteins. [14] However, as far as we know, this is the first report in which the technique has been applied to the transport of inorganic ions. The method has been used to visualize and quantify chloride transport by a powerful anion transporter, and could in principle be applied to many other ion transport processes.…”