Distinct, Phase-Dependent Orientations of GPI and GPI-Anchored Proteins in Lipid Bilayers Revealed Using Spin...

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.