ABSTRACTSTUDY OF DOMAIN SWAPPING OF ILBP FAMILY ByNona EhyaeiIntracellular lipid binding proteins (iLBPs) are small soluble proteins in cytosol responsible for transport of different insoluble hydrophobic molecules. Members of this family have relatively the same structures including ten stranded beta barrel and two alpha helices located at the mouth of the internal binding cavity like a cap for binding pocket of these proteins. Studies that have been done on folding pathway of iLBP reports the early beta barrel formation in the folding pathway for this family of proteins.Human cellular retinol binding protein II (CRBPII) is a member of the iLBP family and is responsible for transport of retinal and our group have been using this protein in an effort to create rhodopsin mimics to create Schiff base with retinal. During studies on hCRBPII for protein design, my former lab mates discovered a domain swapped dimer for this protein. Domain swapping is a process by which two or more monomers exchange an identical part of their structures to form dimer or higher order oligomers. The swapped region in this protein is three beta strands with two alpha helixes which is about half of the protein.Surprisingly, mutational studies on hCRBPII have shown that with single mutation, we can change the ratio of monomer and domain swapped dimer which are folding products in folding pathway of this protein. We tried to find the effect of different residues on domain using ourstrategy called “Phase-problem”. Existence of domain swapping for hCRBPII, can have physiological relevant and may affect the folding pass way for this family of proteins.In the meanwhile, another member of the iLBP family, Fatty acid binding protein 5 (FABP5), has reported to have a very similar domain swapped dimer. This protein is found mostly in epidermal cells, but also in other tissues, such as brain, liver, kidney, lung, and adipose tissue. Existence of domain swapping in Holo FABP5 is very interesting for us, since this protein is in the same family as hCRBPII and we are interested to investigate on the structure of Apo FABP5. In the course of these studies, we resolved two domain swapped structures of FABP5 bind to palmitic acid and studied the melting points of monomer and dimer FABP5 relative to each other.Existence of domain swapping for hCRBPII and FABP5 may lead to allosteric regulation, also huge effect on the folding passway for this family of proteins. Previously, all studies in our lab for domain swapping was through bacterial expression, however, in order to check the physiological relevance of this phenomena we made studies on mammalian expression of hCRBPII. We successfully expressed this protein in HeLa cells and got closer to find the size of this protein in mammalian expression by using size exclusion chromatography and western blotting.
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