Heterologous production and characterisation of cation diffusion facilitator proteins from the marine bacterium Maricaulis maris MCS10

Cation diffusion facilitators are a diverse family of transporters responsible for the efflux of zinc and other divalent metals from cells. Several putative members of this family were selected as potential targets for research with the intent of removing difficult targets as problems arose. Of particular interest was the smallest target, MmCDF3, from Maricaulis maris, due to having no apparent C-terminal cytoplasmic domain, yet seemingly maintaining the tetrahedral zinc-coordinating active site and six transmembrane helices found in all known CDFs. This study focused on the expression, purification, characterisation and crystallisation of members of the CDF family, specifically targets from M. maris. High level expression (6-7 mg/L) and purity has been obtained for the MmCDF1 and MmCDF3 proteins, although both could only be obtained in a heavily aggregated state when purified in DDM. Acquiring CDF proteins in a non-aggregated stable form was necessary for repeatable crystallisation experiments and was accomplished by means of a detergent exchange in the presence of high salt concentrations. MmCDF3 was determined to be stable after an exchange with the detergent FC12, but only into a mixed micelle consisting of both FC12 and DDM. Investigations into the exact nature of the micelle containing stable MmCDF3 were performed by constructing mixed micelles of specific detergent ratios. Determination of function was performed by isothermal titration calorimetry, which showed that MmCDF3 was capable of binding zinc and cadmium ions, although in a much reduced fashion to the larger CDF proteins. This study did highlight a potential mechanism through which MmCDF3 and other smaller CDFs might bind their substrate ions in the cytoplasm. Following successful recovery of the stable homogenous protein, crystallisation was attempted with little success. However, now that a method has been established for obtaining a pure, homogenous and stable CDF protein, further crystallisation studies can be attempted.