AbstractsBiology & Animal Science

ATP-binding cassette (ABC) transporters are ubiquitous multi-subunit transmembrane (TM) protein complexes. They play vital roles in physiological processes like nutrient uptake in prokaryotes, lipid metabolism and extrusion of toxic compounds. ABC transporters hamper chemotherapy of cancer and treatment of infectious diseases by actively removing drugs from the cytoplasm. Both, ABC import and export systems require the energy obtained from ATP hydrolysis within their nucleotide binding 'domains' (NBDs) to translocate their substrates across lipid bilayers. In contrast to exporters, ABC importers rely on an additional substrate binding protein (SBP) that captures substrate molecules and delivers them to the transmembrane domains (TMDs) of the transporter. X-ray structures of the E. coli maltose importer in different nucleotide-bound states and in a lipid-free environment have been published. On this basis, a transport cycle was proposed that includes an ‘Alternating Access’ mechanism describing the structural changes in ABC transporters upon substrate translocation. However, despite the many similarities, functional differences between the two transporter classes exist and the details of the translocation mechanism in the native lipid environment are far from understood. In the frame of this thesis, structurally distinct states of the amino acid importer ArtMP-J from Geobacillus stearothermophilus were investigated in lipid bilayers using ADP and the non-hydrolyzable ATP-analog AMPPCP to capture the post-hydrolysis and transition states, respectively. Magic angle spinning (MAS) solid-state NMR (ssNMR) was used as the primary tool to characterize lipid-embedded ArtMP. The occurrence of non-covalently linked subunits (ArtM and ArtP, two of each) in ArtMP-J facilitated their individual labeling within the functional complex. As a major part of this thesis the protocols for labeling of ArtP or ArtM in ArtMP were improved to yield preparations suitable for subsequent spectroscopic investigations. Heterologous expression of ArtM and its enrichment with NMR-active isotopes had to be optimized and highly cost-efficient 2H,13C,15N labeling of the TMD was achieved by adapting the ‘condensed Single Protein Production’ protocol. The thermodynamic parameters of nucleotide binding to ArtP and ArtMP were analyzed by isothermal titration calorimetry (ITC). Experiments with the soluble NBD ArtP alone yielded comparable affinities for ATP and ADP (6 µM), while the non-hydrolyzable analogs AMPPNP and AMPPCP bound by factors of 8 and 45 weaker. The detergent-solubilized ArtMP complex showed strongest affinity for ADP (13 µM) while both AMPPNP and AMPPCP bound with KDs of around 70 µM. Interestingly, the interaction of ArtMP and ADP was endothermic, while all other nucleotides bound in an exothermic reaction. In the next step, structural effects of nucleotide-binding to ArtP were investigated by solution NMR. Binding of ATP and ADP led to identical chemical shift changes in agreement with the comparable KDs, suggesting very…