Membrane proteins perform in many critical physiological processes, constituting 25 – 30% of prokaryotic and eukaryotic genomes, and represent up to 70% of current drug targets. However, the number of three dimensional structures of membrane proteins elucidated has been modest in comparison with those of soluble proteins, and this is attributable to three major bottlenecks: expression, purification and structure determination of these hydrophobic proteins.
A genomic approach was conducted to overcome these bottlenecks for two families of membrane proteins: (1) the NCS1 family of transporters and; (2) the histidine kinases of two-component signal transduction systems of E. faecalis. The genes encoding these membrane proteins were cloned separately into plasmid pTTQ18-His6 and subjected to expression trials in Escherichia coli. Purification of the recombinant intact proteins from E. coli membranes was undertaken using Ni-NTA chromatography, and for proteins that were produced in sufficient quantities crystallisation trials and investigations of structural characteristics were performed. The activities of membrane-bound and/or purified proteins were also tested, and in the case of the histidine kinases, these activity assays were expanded to successfully identify modulating signals. This is the first time that direct in vitro activity assays using intact proteins have proven successful for identification of environmental ‗signals‘ in enterococci.
In this study, thirteen NCS1 transporters were cloned. Twelve of the thirteen cloned proteins were expressed in E. coli membranes, seven were successfully purified, and substrates transported by five of these were confirmed. Three of these transporters, were further characterised, including; (1) a cytosine transporter – CodB, (2) an allantoin transporter – PucI, and (3) a uracil transporter – PA0443, and their substrate specificities and kinetics were also determined.
All sixteen membrane histidine kinases of E. faecalis were successfully cloned (six were assigned to me in a joint project with Dr Hayley Yuille). Fifteen of the sixteen histidine kinases were successfully expressed in E. coli, and thirteen were purified. Eleven of the fifteen membrane-bound histidine kinases and twelve of the thirteen purified proteins were active in autophosphorylation activity assays. Three histidine kinases were studied further, including; (1) EF3197, which responded to increasing concentrations of a reducing agent, suggesting a possible role in redox-sensing, (2) EF1051, which was further purified by size exclusion chromatography and showed promising results in crystallisation trials for future structural determination, and (3) EF1820 (FsrC), which was activated in in vitro assays by its pheromone signal, gelatinase biosynthesis-activating pheromone (GBAP). This study showed GBAP-induced activity of FsrC was inhibited by the anti-HIV inhibitor, siamycin I, identifying unequivocally and for the first time, the target protein for siamycin I inhibition in the Fsr pathway.
This study presents a successful genomic approach for the production of milligram quantities of membrane proteins leading to characterisation and crystallisation studies. The methods can now be applied to further membrane proteins.
CORE (COnnecting REpositories)
CORE (COnnecting REpositories)