2] Preparation and reconstitution of membrane-associated maltose transporter complex of Escherichia coli

Abstract

This chapter describes an in vitro method for reconstitution of transporter complexes into proteoliposome vesicles. This method is based on the Ambudkar–Maloney modification of the octylglucoside dilution procedure. Such a method is convenient for studying the interaction of maltose-binding protein (MBP) with either wild-type or MBP-independent transporter complexes. MBP-independent transporter complexes hydrolyze adenosine triphosphate (ATP) in the absence of both MBP and maltose, whereas, the wild-type transporter requires the presence of both for ATP hydrolysis to occur. This method allows for kinetic studies of ATP hydrolysis and substrate uptake. Maltose and its higher homologs—the maltodextrins—are actively transported across the cytoplasmic membrane of Escherichia coli via a high affinity, periplasmic binding protein-dependent transport system. In wild-type cells, MBP is required for substrate transport across the membrane.