| Located on the inner membrane of Oxalobacter formigenes , the oxalate transporter (OxIT) is responsible for breaking down oxalate into formate and carbon dioxide. This cycle in turn generates an electro chemical gradient across the membrane, which is utilized by F0F 1-ATPase for energy production. OxIT belongs to the Major Facilitator Superfamily (MFS); a large transporter family involves in transporting a variety of substrates. Since this family is highly conserved, and members are considered to share common architecture and transporting mechanism, we believe that the study of OxIT will shed light on the understanding of the function and mechanism of other family members, including the more clinically relevant ones.;My PhD thesis is devoted to two aspects of studies, OxIT oligomeric state determination and crystallization for X-ray crystallographic study. In chapter two, we measured the core protein molecular mass with the triple-detector size-exclusion chromatography (TD-SEC); probed the inter-molecular distance with cysteine and lysine-mediated crosslinking experiments; and finally assessed the minimal functional unit with kinetic study. All data uniformly agreed that OxIT is a functional monomer both in detergent solution and membrane bilayer.;In chapter three, we described our tortuous journey towards the three-dimensional crystallization of OxIT. After years of effort and strategic screening of thousands conditions, we were able to generate crystals with maximum diameter of 100 mum. In addition, we developed a fast and reliable method for stabilization test, created fusion protein constructs with more hydrophilic area, and selected out single cysteine derivatives with excellent stability. More importantly, we have accumulated sufficient amount of data to better aid future trials. We believe, with the rapid development of X-ray crystallographic technique and the collaboration with Hauptman-Woodward Membrane Protein Screen Center, the determination of OxIT structure is very feasible. |
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