ABC proteins or traffic ATPases are members of a large superfamily of translocators, both prokaryotic and eukaryotic. The eukaryotic members include the cystic fibrosis transmembrane regulator (CFTR), the P-glycoprotein of multidrug resistance (MDR), the gene responsible for adrenoleukodistrophy (ALD), the heterodimeric transporter associated with antigen processing (Tap1/Tap2), the transporter involved in Stargard macular dystrophy, and many others of medical importance. Among the prokaryotic members are bacterial periplasmic permeases, which have been extensively characterized. Considering the extensive homology between all of these proteins it is likely that there will also be strong similarities between eukaryotic and prokaryotic systems in their mechanism of action. Thus, prokaryotic permeases provide excellent models for achieving a general understanding of the activity and function of these translocators. The proposed work aims at understanding in detail the 3-dimensional structure of a model system: the histidine permease. We plan: 1). The spatial organization of the permease will be assessed by chemical cross-linking, NMR, and spin-labeling studies which will provide information on the three-dimensional structure by defining the nearest neighbor contact between the individual components. We will purify the individual cross-linked products, perform proteolytic digestion, HPLC separation of the peptides and mass spectrometry analysis. Comparison between patterns obtained from cross-linked products and from untreated complex will indicate the sites of contact. Experiments will also be performed on the permease in various stages of its activity cycle. 2). We will also try and obtain the crystal structure of the entire permease. If successful, this would provide the best description of the structure. 3). Crystal structures will be obtained of various mutants of the separated ATP-binding domains (HisP), which will provide information on which signals are needed to initiate ATP hydrolysis and on signaling mechanisms. Examination of these and other aspects of the histidine periplasmic permease will contribute to the understanding of general mechanistic questions for these transporters and will help unravel how their several sub-domains function. An understanding of the structure of these systems is essential for developing new approaches for investigating eukaryotic transporters. This knowledge can then be applied to help develop therapeutic tools for human diseases.
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