This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The steroidogenic acute regulatory protein (StAR) facilitates the rapid movement of cholesterol into adrenal and gonadal mitochondria, where cholesterol becomes the substrate for the first step in the synthesis of all steroid hormones. StAR works out the mitochondrial membrane required for the stress responses and for reproduction. The C-terminus of StAR is resistant to proteolysis in ther presence of liposomes, implying that this region is associated with the membrane.Crystal structures are available for two proteins closely related to StAR, MLN64 and StARD4, revealing that two loops are adjacent to the C-terminus. Conformational changes of the loops could pivot the C-terminal helix and open the roof to allow cholesterol to bind to the protein. The two loops contain negative charged residues, while the C-terminus is rich is positively charged residues, suggesting that salt bridges and hydrogen bonds may stabilize the structure. The hydrophobicity of the surface surrounding the loops suggests that the loop could be the membrane docking site for lipid loading and unloading.We plan to determine whether the negatively charged residues in the loop regions play a role in substrate binding and membrane association. First, are building a human StAR protein model using the crystal structures of the closely related proteins MLN64 and StARD4, using the 'Chimera' program developed in the Computer Graphics Lab. From the model, we will measure the side-chain distances between the negatively charged residues from the loop regions and the positively charged residues from C-terminus. We will then mutate these residue(s) in silico and evaluate their effects on the protein geometry and conformation. Potentially heuristic mutants will then be built and studied in vivo at the bench.
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