The maltose transporter is a member of the ABC (ATP-binding cassette) superfamily, which has highly conserved sequences for their ATP binding domains and has characteristic transmembrane domains that form the transport pathway. The superfamily includes transporters in prokaryotic and eukaryotic cell systems. Many of the transporters have been demonstrated to be critical in normal cell function, and defects in these proteins have been related to a number of diseases, such as cystic fibrosis. The maltose transporter is found in the cytoplasmic membrane of E. coli and has its maltose transport processes coupled to the ATP hydrolysis. The transporter is a multi-subunit protein complex consisting of three different polypeptides: one MalG, one MalF and two MalK. The maltose transporter has previously been purified in small quantities. We have developed a new purification method that yields a quantity of a few milligrams of maltose transporter in a fully active form. In this proposed research, we will further improve our purification of this membrane protein in a homogeneous state using additional chromatographic columns. With the purified protein, we will perform 2-dimensional crystallization of this protein for electron crystallographic structure determination. We will use the crystallization techniques involving reconstitution of the purified protein with selected lipids that we and other have developed to obtain 2-dimensional crystalline patches. When the crystals become available we will first determine the low resolution structure of the transporter using the 3-dimensional reconstruction method in electron microscopy to provide the overall architecture of the transporter. Subsequently we will obtain the high resolution structure by high resolution electron diffraction and imaging methods to provide the molecular basis for understanding the functional mechanism of the maltose transporter. This understanding is expected to reveal the general principles governing the molecular mechanism of the ABC superfamily and to provide insight into the structural basis for a number of diseases.
