| Summary: | Nucleoside transporters catalyse the passage across the cell membrane both of natural nucleosides, which play key roles in cell physiology, and of nucleoside analogues, which are widely used in chemotherapy. In humans two structurally unrelated families of nucleoside transporters have been identified, the equilibrative and concentrative nucleoside transporters (hENTs and hCNTs, respectively). A bacterial homologue of the hCNTs, the Escherichia coli transporter NupC, has been used as a model system for studies of the structure and function of the CNT family. It has an unusual topology with both the N- and C-termini in the periplasm, which precludes the addition, at these termini, of many conventional tags that aid in purification. Therefore, different tagging methods have been investigated, leading to the discovery that inserting a hexahistidine tag in the putative central cytoplasmic loop of Nu pC generates a construct that can be overexpressed in the E. coli membrane and in which transport activity has been retained. NupC can also be successfully purified using this histidine tag and purification conditions have been optimised. Methods of enhancing the mono- dispersity of the purified protein have been investigated in order to produce material suitable for 3-D crystallisation trials and other structural and functional analyses. 3-D crystallisation trials were carried out, which yielded crystals in a number of different conditions, signifying substantial progress towards the elucidation of a NupC crystal structure. Due to the current absence of a crystal structure, thiol cross-linking investigations of Nu pC were carried out in order to examine the relative distances between pairs of introduced cysteine residues and thereby allow the prediction of a 3-D model of Nu pC. A successful protocol for investigation of distances between putative intramembraneous positions has been established, although no cross-linking between the T176 and S354 positions investigated was observed. In addition, purification and thiol cross-linking of internally Hiss-tagged NupC suggested that it was oligomeric and therefore preliminary investigations into its quaternary structure were performed. Glutaraldehyde cross-linking and native gel electrophoresis suggested that purified NupC may be present as a trimer, as well as other larger oligomers. Cysteine mutants of a number of residues in putative transmembrane domains (TMs) five and nine were used to analyse the role of each position in NupC structure and function. The results suggested that residues in TM5 may play an important role in NupC function and at least in part contributes to the permeant translocation pathway. 4 Results suggested that TM9 lines the translocation pathway as transport activity for three mutants in this helix was shown to be inhibited by thiol reagents. These findings correlate with similar studies in hCNT3, suggesting that NupC shares functional similarities with the human transporters and therefore confirming it as a good model for this family. In addition a protocol, which uses a fluorescent thiol reagent, was established for examining permeant protection of thiol reagent binding to purified cysteine mutants of Nu pC, although no protection was seen for the mutants, T176C and S354C, which were investigated in this study.
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