Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion

In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmemb rane proteins at the molecular level. Furthermore, reconstitution in this m anner would also eliminate potential artifacts arising from secondary inter actions of proteins, when reconstituted in planar membranes supported on so lid surfaces. However, assembly procedures of GUV preclude direct reconstit ution. Here, for the first time, a method is described that allows the cont rolled incorporation of membrane proteins into GUV. We demonstrate that lar ge unilamellar vesicles (LUV, diameter 0.1 mum), to which the small fusogen ic peptide WAE has been covalently attached, readily fuse with GUV, as reve aled by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devi sed. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confoca l imaging intensity revealed that similar to6 to 10 LUV fused per mum(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dyalisis. BR did not affect GUV-LUV fusion and the protein was stably inserted into t he GUV and functionally active. Fluorescence correlation spectroscopy exper iments show that BR inserted into GUV undergoes unrestricted Brownian motio n with a diffusion coefficient of 1.2 mum(2)/s. The current procedure offer s new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state.