Approaches for preparation and biophysical characterization of transmembrane β-barrels

Abstract

Membrane proteins are influential members of the cellular machinery, as they are involved in a range of critical functions from immune receptors and ion transporters to enzymes and signaling molecules. These facets render membrane proteins as ideal drug targets. Despite their significance in biological systems, our knowledge of the structural and functional behavior of membrane proteins remains considerably limited, when compared with their soluble counterparts. This limitation continues to persist, owing largely to the difficulties in membrane protein purification and amenable methods for their characterization. In this review, we outline the methods for the extraction and purification of transmembrane β-barrel proteins, which will serve as a guide for generating membrane protein preparations in high amounts and of sufficient purity. Next, we provide a detailed description of the currently available protocols for efficient refolding of β-barrel membrane proteins in both lipidic and detergent systems. These protocols are designed to enable the protein to attain its native structural and functional characteristics in any chosen membrane mimetic environment. Further, we detail the current methodologies being employed for the biophysical and structural characterization of transmembrane β-barrels, using illustrative examples drawn from recent studies using various β-barrel membrane proteins. This consolidated summary of protocols and strategies will prove highly useful to membrane biologists and can be applied to the study of any uncharacterized β-barrel membrane protein.