Triangulating a Tightly Bound Lipid on a Membrane Protein by Paramagnetic Solid-State NMR

Nonannular lipids tightly associate with membrane proteins, influencing their structure and function. Here, we present a solid-state NMR (ssNMR)-based strategy to localize such lipids on protein surfaces with a high specificity. We previously identified a tightly bound glycophospholipid which copurifies with <i>Anabaena</i> Sensory Rhodopsin trimers and displays resolved NMR signals. By introducing paramagnetic labels at defined sites and measuring paramagnetic relaxation enhancements (PREs), we triangulate the lipid's position near a periplasmic intermonomer cleft. Docking calculations guided by PRE restraints further define a nonannular lipid binding site. Our approach provides a broadly applicable framework for mapping tightly bound lipids in membrane proteins under native-like conditions.