The focus of this study is proteins from the ArAE family, specifically two subfamilies: firstly, the ALMT family of plant membrane channels, which have numerous vital roles in plants, and secondly, the bacterial family first described as PET inner membrane exporters. Constructs were created for expression of firstly, the C-terminal domain (CTD) of wheat ALMT1 in E. coli and secondly,
three full-length ALMTs (ALMT from wheat and ALMT5, ALMT9 from Arabidopsis) in Nicotiana benthamiana for structural and biochemical studies.
Unfortunately, it appears that the CTD is not an independent soluble domain as
originally thought, and this domain is now predicted to contain transmembrane
helices, making it unsuitable for study in the manner planned. Similarly,
production of full length ALMTs was unsuccessful as when extraction and
purification was attempted, the protein degraded. Thirdly, a bacterial member of
the ArAE family was expressed and characterised: AaeB from E. coli, along with
its putative binding partner, AaeA. This was shown to form a complex with and
be vital for the stability of AaeB. A strategy was devised for expression,
solubilisation, and purification of these proteins and once they were obtained in
pure form they were subjected to a range of biochemical and structural
experiments. Microcrystals of AaeA were produced, towards a strategy for
structural determination by X-ray crystallography. The first Electron Microscopy
examination was performed on complexes of AaeB, and negative stain classes
were produced. The first in silico homology model of AaeA has been produced
and validated by CD spectroscopy, providing a range of insights. The complex
formed by AaeA and AaeB has also been probed by crosslinking and SEC
MALLS analysis, suggesting a 6:2 or 6:3 stoichiometry. Together this has
furthered our understanding of this poorly characterised membrane protein
family and provided a set of clones and protocols for future studies.
