Determination of membrane protein orientation upon liposomal reconstitution down to the single vesicle level

Abstract

Reconstitution of membrane proteins into liposomal membranes represents a key technique in enabling functional analysis under well-defined conditions. In this review, we provide a brief introduction to selected methods that have been developed to determine membrane protein orientation after reconstitution in liposomes, including approaches based on proteolytic digestion with proteases, site-specific labeling, fluorescence quenching and activity assays. In addition, we briefly highlight new strategies based on single vesicle analysis to address the problem of sample heterogeneity.

Reconstitution of ATP-dependent lipid transporters: gaining insight into molecular characteristics, regulation, and mechanisms

“…Membrane proteins reconstituted in liposomal model membrane systems have either the cytosolic side (inside-out orientation) or the extracellular side (outside-out orientation) exposed. ATP-dependent transporters appear to prefer an inside-out orientation in the vesicle, due to bulky domains and the curvature of the vesicle [ 78 ] but the exact protein orientation is hardly predictable, which limits the quantitative and functional analysis of vesicle preparations [ 79 , 80 ].…”