CRYSTALLIZATION OF AN ABC TRANSPORTER

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Maltose transporter is a member of the ABC (ATP-binding cassette) superfamily, responsible for ATP coupled transport of maltose across E. coli cytoplasmic membranes. Members of the superfamily can be found in prokaryotic as well as eukaryotic cell systems. Many of these transporters have been demonstrated to be critical for normal cell functions, and defects in these proteins have been related to a number of diseases such as cystic fibrosis. Maltose transporter is a large multi-subunit protein complex formed by three different polypeptides, consisting of one MalG, one MalF and two MalK. The applicant has developed a purification method that yields milligram quantities of active maltose. The current proposal is to determine the crystallization conditions of maltose transporter in order to produce crystals that are suitable for subsequent crystallographic structure determination. This will require (1) characterization of the protein in various detergents, (2) production of homogeneous maltose transporter in milligram quantities selected detergents, (3) the study of solution phase behavior of the detergent in polyethylene glycols and other precipitating agents, (4) the crystallization trials of the protein in selected detergents using the crystallization matrice typically associated with the crystallization of water-soluble proteins, and (5) the refinement of crystallization conditions using additives such amphiphiles, divalent cations and other detergents that have a high critical micellar concentration. Crystals will be evaluated for diffraction quality and tested with various cryo-protectants for cryo- crystallography. The goal is to obtain high quality crystals and to develop flash-freezing conditions for subsequent high resolution structure determination in order to provide the molecular basis for understanding the functional mechanism of maltose transporter. This structure should reveal general principles governing the molecular mechanisms of members of the ABC superfamily, and provide insight into the structural basis of diseases associated with defects in the transporters.