Distinct, Phase-Dependent Orientations of GPI and GPI-Anchored Proteins in Lipid Bilayers Revealed Using Spin Labeling and Power Saturation Electron Paramagnetic Resonance Depth Profiling

Glycosylphosphatidylinositols (GPIs) help anchor and organize surface proteins, while the interfacial conformations of GPIs and GPI-anchored proteins (GPI-APs) on cells, which determine their functions, remain unclear. In this work, a bifunctional GPI anchor bearing clickable azide and biotin─an affinity tag─was used to access labeled GPI anchors and GPI-AP analogs. Employing spin-labeled GPI and GPI-streptavidin conjugate as probes, together with spin-labeled “ruler” lipids and power saturation electron paramagnetic resonance (EPR) profiling, we quantified their accessibilities to membrane-tethered and soluble spin relaxants, with the probes and rulers incorporated in 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) and dipalmitoylphosphatidylcholine (DPPC) liposomes. It is shown that GPI-streptavidin adopts a “flop-down” conformation toward the lipid membrane in POPC but an upright “lollipop” state in DPPC. Conversely, free GPI flips between these two conformations, although the equilibrium is also lipid phase-dependent. Overall, in ordered membranes, protein binding pulls GPI away from the bilayer and biases it toward an extended conformation. Thus, this study establishes a general synthetic and EPR spectroscopic framework for mapping the orientations and conformational dynamics of GPIs and GPI-APs in bilayers and reveals how GPI anchors and lipid phases control the organization and presentation of GPI-APs in membranes, thereby regulating their binding, signaling, and other biological activities.