| Summary: | Sodium-proton (Na+/H+) antiporters are secondary membrane protein transporters present in all living cells and are critical for sodium, pH and cell volume homeostasis. Their deregulation of transport activity has been linked to human diseases, such as, hypertension, heart failure and epilepsy and consequently they may be targets for drugs. In 2005, the first crystal structure of a Na+/H+ antiporter, NhaA from Escherichia coli, was solved at 3.45 Å resolution in an inward-facing conformation at pH 4 where the protein is inactive. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH and is active above pH 6.5. The main goal of this thesis was to solve a crystal structure of a Na+ /H+ antiporter in an active state. One of the problems in producing milligram quantities of purified membrane protein for crystallography is poor overexpression. For this reason, we first sought to improve membrane protein overexpression by developing a new expression platform, which we have called 'MemStar' in E. coli using a test-case of control proteins, overall showing a boost in expression levels to at least 12 mg.L-1. This thesis describes the crystal structure of NapA from Thermus thermophilus, an NhaA homologue, which was solved to 3 Å in an outward-facing conformation at pH 7.8 in an active state. This NhaA homologue was selected as purified protein could grow better diffracting crystals than NhaA. The stability of NapA was also more suitable for purification in a small micelle detergent to improve diffracting resolution. Although NhaA crystals did not form above pH 6.5, a stabilised mutant was useful to confirm the position of a critical residue important in the mechanism. Structural comparisons with the NapA structure show the core domain moving relative to the dimerisation domain, similar to a rocking bundle model observed in other structures of different secondary active transporters sharing conserved structural features in their membrane protein folds also present in NhaA and NapA. This work has provided us with a fresh insight into the mechanism of Na+/H+ antiporters.
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