Structure and Function Studies of Bacterial Membrane Transport Proteins

Nucleoside transporters play key roles in the uptake of nucleosides for salvage pathways of nucleotide synthesis in many organisms. In mammals, they also influence physiological processes, such as cardiovascular activity, via regulation of extracellular adenosine concentrations, and are the route of uptake for many anticancer and antiviral drugs. Unfortunately, understanding their molecular mechanisms in humans is hampered by their low abundance in the membrane and difficulty of expression. To obviate these problems, in the present project, bacterial proteins from the Hydroxy/Aromatic Amino Acid Permease family were studied as potentially more amenable models of the human Equilibrative Nucleoside Transporter family of proteins for structure/function analysis using bioinformatic approaches including molecular modelling. Although these families show little clear sequence similarity, they share a predicted 11 transmembrane topology, with a cytoplasmic N-terminal. In parallel, attempts were made to overexpress an Hi\AAP family member for structural studies. The present project also aimed to gain an understanding of the molecular mechanisms of transport by investigating the Escherichia coli nucleoside/proton co-transporter NupG. This member of the ubiquitous Major Facilitator Superfamily of transporters provides an excellent system for structure/function studies because it can be expressed at very high levels in the. bacterium and subsequently purified in a functional form. Moreover, a molecular model of the protein, based on its distant homology to the E. coli lactose transporter, was developed in this study. To investigate structure/function relationships in NupG, residues predicted from the model to be functionally important or to be located in the vicinity of the nucleoside-binding site were mutated to cysteine or other amino acids. Analysis of the kinetic properties of the mutants, and their susceptibility to thiol reagents where appropriate, has provided considerable information on the identities of residues likely to be involved in permeant recognition and translocation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo