Browsing by Author "Mahmood, Hamid"

Nucleosides are one of the fundamental building blocks of the cell and are precursors of DNA and RNA and serve as universal signalling molecules for cell surface receptors. These nucleosides play vital roles in myriad of physiological processes, such as cardioprotection, platelet aggregation and coronary vasodilation. In clinical settings, synthetic analogs of nucleosides are used as therapeutic against cancer and other viral diseases. In human, uptake and regulation of nucleosides and their analog drugs is facilitated by the two families solute carrier membrane transporter proteins (SLC); SLC28 and SLC29. SLC29 consists of four human equilibrative nucleoside transporter members (hENT1-4) with different sub-cellular localization, tissue distribution and substrate selectivities. Human equilibrative nucleoside transporter 1 (hENT1) is the major plasma membrane nucleoside transporter and has shown to play an important role in adenosine signalling and anticancer therapies. Despite the significant physiological and pathophysiological role, little is known about the structure and molecular function of ENT homologs. Structural studies of hENT1 and other nucleoside transporter is hampered by their low expression and hydrophobic nature. The aim of this thesis work is to find suitable nanobodies (Nbs) that may serve as crystallization chaperone to facilitate the structural studies of hENT1 transporter. Camelid heavy chain only antibodies (Nanobodies) raised against recombinant hENT1 were screened for their suitability for future structural investigation of hENT1. Selected nanobodies were expressed and purified from E.coli cell in milligram quantities. Affinity of nanobodies with hENT1 was assessed using co-elution on size exclusion chromatography. High affinity binders were further screened for their ability to conformationally stabilize hENT1. In future, high affinity nanobodies will be explored for x-ray crystallization and single particle cryo-electron microscopy of hENT1. For cryo-EM it's important to convert the selected Nbs into megabody which constitute large scaffold proteins. 3D structure determination of hENT1 will be significant in understanding its molecular function and to accelerate rational drug designing against cancer, HIV and other viral infections.