Biophysical and functional properties of lipid-modified proteins and model lipopeptides

Three related studies were carried out to understand better the physical properties, cellular function, and means to prepare artificially proteins 'anchored' to membranes by two hydrophobic chains.The first study entailed the development of efficient methodologies to conjugate antibody Fab$ sp prime$ fragments to suitable lipid 'anchors' in liposomes, to allow for the targeted delivery of liposome-associated materials to cells expressing the appropriate cell surface determinant. To monitor the production of Fab$ sp prime$ fragments bearing reactive thiol groups, we developed a maleimide-polyethylene glycol (PEG) conjugate 'thiol reagent' for use in an SDS-PAGE mobility shift assay. Optimizing conditions for Fab$ sp prime$ preparation developed by this approach, we achieved efficient and reproducible coupling of Fab$ sp prime$ fragments to liposomes via a novel lipid 'anchor'. Such Fab$ sp prime$-'targeted' liposomes efficiently delivered encapsulated materials to cultured cells in vitro.The second study used lipid-modified peptides to investigate the strength of membrane anchoring conferred by dual lipid modifications of the types attached to various intracellular proteins. Based on kinetic measurements of interbilayer transfer of such peptides and peptide-macromolecule conjugates, we suggest that doubly-modified proteins exhibit extremely slow rates of spontaneous intermembrane transfer. We have proposed mechanisms by which this property of 'dual-anchored' proteins may be exploited to achieve efficient subcellular targeting by 'trapping' mechanisms.The third study sought to assess current models of the mechanism of preferential localization of glycosylphosphatidylinositol (GPI)-proteins to specialized plasma membrane invaginations known as caveolae. This was accomplished using cell membrane-incorporated lipid-PEG-biotin-streptavidin conjugates, whose lateral distribution under various conditions ($ pm$ crosslinking) shows significant overlap with that of endogenous GPI-proteins. We propose a novel mechanistic model based on 'surface-crowding' effects, for the preferential association of lipid-anchored molecules with caveolae, particularly upon crosslinking