| The topic of my doctoral dissertation is probing the structural features of caveolin-1. Caveolin-1 is responsible for the formation of caveolae which are 50-100 nm diameter invaginations in cell membrane. The unique feature of caveolin-1 is the transmembrane domain which has a horseshoe conformation where both the N- and C- termini are facing the cytoplasm and the polypeptide chain turns inside the lipid bilayer. In Chapter 1, expression and purification methods are discussed in order to obtain high yield of hydrophobic proteins. Trp Leader fusion system was employed to send toxic membrane proteins into the inclusion bodies to protect the cells. Also, optimization of purification methods are discussed to obtain high purity membrane proteins, especially caveolin-1 constructs. In Chapter 2, the horseshoe topology of caveolin-1 transmembrane domain (96-136) was investigated. Caveolin-1(96-136) displays a helix-break-helix motif and G108, I109, and P110 were responsible for the break region which is a putative turn. Various point mutations were performed to give insight into these three residues. In Chapter 3, the caveolin-1 P132L mutation was probed. The P132L mutation is a naturally occurring point mutation that is found in 16% of breast cancer patients. Comparison of the wild-type and P132L mutation shows an extension of the secondary helix by 4 amino acids. In Chapter 4, a new construct containing both the scaffolding domain and the transmembrane domain was studied to figure out whether the scaffolding domain is part of the transmembrane domain or a separate domain. CSI analysis of caveolin-1(82-136) shows that the scaffolding domain is an extension of the first helix in the transmembrane domain which suggests that the scaffolding domain is not a separate domain. In Chapter 5 various methods of incorporation of caveolin-1 constructs into bicelles and application of bicelles are investigated. Various methods were able to incorporate small amount of caveolin-1 constructs into the bicelles but not at a concentration for NMR studies. Moreover, bicelles were used as templating medium to form periodic meso-porous material. |
