Protein-protein interactions and conformational changes of the maltose ABC transporter of Salmonella typhimurium II

The family of ABC transport systems comprises a diverse class of transport proteins that couple the translocation of solutes across biological membranes to the free energy of ATP hydrolysis. Members of the family exist in all organisms and many of these proteins are of considerable medical importance. ABC transporters share a common architectural organization comprising two hydrophobic transmembrane domains (TMDs) that form the translocation pathway and two hydrophilic nucleotide binding (ABC) domains (NBDs) that hydrolyze ATP.he conformational changes in the NBDs induced by ATP binding/hydrolysis, and the means by which they are transmitted to the TMDs to effect substrate translocation, remain largely to be elucidated. Using the well-studied binding protein-dependent maltose ABC transporter of Salmonella typhimurium (MalEFGK2) as a model system, we aim to define sites of domain interactions in greater detail and to identify residues that are involved in signalling events during the transport cycle. Experimental tools to achieve these goals will include synthetic peptide libraries to be employed in binding studies as well as unspecific and site-specific crosslinkers. These experiments will be complemented by studying the consequences of intersubunit crosslinking on maltose transport in vivo and by analyzing the effects of synthetic peptides on complex reassembly in proteoliposomes. Based on these and previous results, we will purify selected cysteine variants of the transport complex and monitor conformational changes during the transport cycle in detergent solution and in proteoliposomes by fluorescence and electron paramagnetic spin resonance spectroscopy.