Synthetic analogues of glycosylphosphatidylinositol-anchored proteins and their behavior in supported lipid bilayers.
Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified proteins in the outer leaflet of the plasma membrane. GPI-anchored proteins play vital roles in signal transduction, the vertebrate immune response, and the pathobiology of trypanosomal parasites. While many GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. We synthesized a series of GPI-protein analogues bearing modified anchor structures that were designed to dissect the contribution of various glycan components to the GPI-protein's membrane behavior. These anchor analogues were similar in length to native GPI anchors and included mimics of the native structure's three domains. A combination of expressed protein ligation and native chemical ligation was used to attach these analogues to the green fluorescent protein (GFP). These modified GFPs were incorporated in supported lipid bilayers, and their mobilities were analyzed using fluorescence correlation spectroscopy. The data from these experiments suggest that the GPI anchor is more than a simple membrane-anchoring device; it also may prevent transient interactions between the attached protein and the underlying lipid bilayer, thereby permitting rapid diffusion in the bilayer. The ability to generate chemically defined analogues of GPI-anchored proteins is an important step toward elucidating the molecular functions of this interesting post-translational modification.
Paulick
Margot G
MG
Department of Chemistry, University of California, Berkeley, California 94720, USA.
Wise
Amber R
AR
Forstner
Martin B
MB
Groves
Jay T
JT
Bertozzi
Carolyn R
CR
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D017261:Glycosylphosphatidylinositols / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D010957:Plasmids; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics
D017261:Glycosylphosphatidylinositols; D008051:Lipid Bilayers; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins