Analysis of Transmembrane β-Barrel Proteins by Native and Semi-native Polyacrylamide Gel Electrophoresis.

Abstract

Membrane-embedded β-barrel proteins are important regulators of the outer membrane permeability barrier of Gram-negative bacteria. β-barrels are highly structured domains formed by a series of antiparallel β-strands. Each β-strand is locked in position by hydrogen bonds between its polypeptide backbone andthose of thetwo neighbouring strands in the barrel structure. Some transmembraneβ-barrel proteins form larger homo- or hetero-multimeric complexes that accomplish specific functions. In this chapter, we describe native and semi-native polyacrylamide gel electrophoresis (PAGE) methods to characterize the organization of transmembraneβ-barrel proteins. We illustrate blue native (BN)-PAGE as an analytical method to assess the formation of protein complexes. Furthermore, we describe a heat-modifiability assay via semi-native PAGE as arapid method to investigate the folding of transmembrane β-barrels.