Investigating Detergent and Lipid Systems for the Study of Membrane Protein Interactions: Characterizing Caveolin Oligomerization.

摘要

Membrane proteins represent an important class of proteins that closely associate or reside within the plasma membrane of the cell. They play a multitude of roles in cell function such as signaling, trafficking, and recently discovered, scaffolding and shaping of the plasma membrane itself. For example, caveolin is a membrane protein that is believed to have the ability to curve the plasma membrane forming invaginations that serve as signaling platforms called caveolae. The curvature of the plasma membrane is believed to be a result of caveolin oligomerization. Caveolin oligomerization was characterized using sedimentation equilibrium analytical ultracentrifugation. Due to the extremely hydrophobic nature of caveolin it was necessary to explore different detergents and lipid systems that support membrane protein structure and function. Not all detergents are conducive to studies of membrane proteins and it is often necessary to determine empirically the best detergent / lipid mimic best suited for biophysical studies. One membrane mimic that has been well-characterized and used successfully to study membrane proteins are bicelles. Bicelles are discoidal phospholipid structures comprised of a long-chain and short-chain phospholipid, typically 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl- sn-glycero-3-phosphocholine (DHPC), respectively. Bicelles provide a true bilayer environment in which to study membrane protein structure and function. These lipid structures were successfully density matched using the method of sedimentation equilibrium in the analytical ultracentrifuge by adding 71.7% D2O as a density modifier. We explored the utility of bicelles as a medium for studying membrane protein interactions in the analytical ultracentrifuge (AUC) by investigating the interactions of caveolin-1. The results of this work show that caveolin-1 does not have the capacity to oligomerize in detergent micelles or in a bilayer environment (bicelles). On the other hand, a naturally-occuring breast cancer mutant, P132L, forms a strong dimer in detergent micelles. A close investigation of the mutant reveals that an extension of helix 2 in the intramembrane region of the protein where dimerization was shown to occur may play a key role in the dimerization of the mutant.;An alternative bicelle system was also investigated using pentaethylene glycol monooctyl ether (C8E5) instead of DHPC to form the rim of the bicelle. The C8E5 / DMPC lipid aggregates were density matched and their properties were characterized using 31P-phosphorus NMR to assess the heterogeneity of the lipid / detergent arrangement, which confirms a bicellar-like arrangement. C8E 5 has a density similar to water (1.007 g / mL) and was shown to form lipid aggregate structures with DMPC that are less dense and require significantly lower quantity of D2O to density match in the AUC making them better suited to the study of membrane protein interactions of small peptides.