9722264 Oakley Lipids are crucial components of many signal transduction pathways, specifically binding to target proteins and modulating their functions. In spite of the biological importance of these interactions, the origin of specificity in protein-lipid interactions is not well understood. This work will focus on the interaction between the actin-binding protein profilin and the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Binding by PIP2 to profilin is both specific and multivalent, with approximately five molecules of PIP2 binding to each molecule of protein. To identify the determinants of specificity in the complex between profilin and PIP2 lipid assemblies, a branched, polymeric, covalent scaffold capable of presenting multiple copies of the headgroup from PIP2 will be used. PIP2 headgroups and headgroup analogs will be attached to polymers of various sizes. The effects on complex formation of the number of headgroups available for binding, the structure of the headgroups attached, and their spatial arrangement will be tested. Furthermore, dendrimeric analogs containing spectroscopic probes of crosslinking moieties will be used to probe the protein binding site. Such analogs will not only allow an exploration of the protein and lipid determinants of specificity, but will also be used to investigate the mechanism by which PIP2 modulates profilin function. The cytoskeleton is a dynamic network of protein filaments that determines the shape of eukaryotic cells and can reorganize to allow directed cell motion in response to external stimuli. The means by which external stimuli are transmitted to the cytoskeleton are incompletely understood. Nonetheless, the lipid phosphatidylinositol 4-5-bisphosphate (PIP2) is known to play a key role, binding to proteins that regulate filament formation in the cytoskeleton and modulating their functions. The purpose of this work is to gain a detailed understanding of the specificity of these protein-lipid interactions, providing too ls for further dissection of cytoskeletal signaling pathways.
